Methods for improving seeds

ABSTRACT

The invention provides methods of modulating seed mass and other traits in plants. The methods involve producing transgenic plants comprising a recombinant expression cassette containing an ADC nucleic acid linked to a plant promoter.

REFERENCE TO RELATED APPLICATIONS

This is a continuation in part of copending application U.S. Ser. No.08/879,827, filed Jun. 20, 1997, which is a continuation in part of U.S.Ser. No. 08/700,152, filed Aug. 20, 1996, now U.S. Pat. No. 5,994,622both of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to plant genetic engineering. Inparticular, it relates to new methods for modulating mass and otherproperties of plant seeds.

BACKGROUND OF THE INVENTION

The pattern of flower development is controlled by the floral meristem,a complex tissue whose cells give rise to the different organ systems ofthe flower. Genetic and molecular studies have defined an evolutionarilyconserved network of genes that control floral meristem identity andfloral organ development in Arabidopsis, snapdragon, and other plantspecies (see, e.g., Coen and Carpenter, Plant Cell 5:1175-1181 (1993)and Okamuro et al., Plant Cell 5:1183-1193 (1993)). In Arabidopsis, afloral homeotic gene APETALA2 (AP2) controls three critical aspects offlower ontogeny--the establishment of the floral meristem (Irish andSussex, Plant Cell 2:741-753 (1990); Huala and Sussex, Plant Cell4:901-913 (1992); Bowman et al., Development 119:721-743 (1993); Schultzand Haughn, Development 119:745-765 (1993); Shannon and Meeks-Wagner,Plant Cell 5:639-655 (1993)), the specification of floral organ identity(Komaki et al., Development 104:195-203 (1988)); Bowman et al., PlantCell 1:37-52 (1989); Kunst et al., Plant Cell 1:1195-1208 (1989)), andthe temporal and spatial regulation of floral homeotic gene expression(Bowman et al., Plant Cell 3:749-758 (1991); Drews et al., Cell65:91-1002 (1991)).

One early function of AP2 during flower development is to promote theestablishment of the floral meristem. AP2 performs this function incooperation with at least three other floral meristem genes, APETALA1(AP1), LEAFY (LFY), and CAULIFLOWER (CAL) (Irish and Sussex (1990);Bowman, Flowering Newsletter 14:7-19 (1992); Huala and Sussex (1992);Bowman et al., (1993); Schultz and Haughn, (1993); Shannon andMeeks-Wagner, (1993)). A second function of AP2 is to regulate floralorgan development. In Arabidopsis, the floral meristem produces fourconcentric rings or whorls of floral organs--sepals, petals, stamens,and carpels. In weak, partial loss-of-function ap2 mutants, sepals arehomeotically transformed into leaves, and petals are transformed intopollen-producing stamenoid organs (Bowman et al., Development 112:1-20(1991)). By contrast, in strong ap2 mutants, sepals are transformed intoovule-bearing carpels, petal development is suppressed, the number ofstamens is reduced, and carpel fusion is often defective (Bowman et al.,(1991)). Finally, the effects of ap2 on floral organ development are inpart a result of a third function of AP2, which is to directly orindirectly regulate the expression of several flower-specific homeoticregulatory genes (Bowman et al., Plant Cell 3:749-758 (1991); Drews etal., Cell 65:91-1002 (1991); Jack et al. Cell 68:683-697 (1992); Mandelet al. Cell 71: 133-143 (1992)).

Clearly, Ap2 plays a critical role in the regulation of Arabidopsisflower development. Yet, little is known about how it carries out itsfunctions at the cellular and molecular levels. A spatial andcombinatorial model has been proposed to explain the role of AP2 andother floral homeotic genes in the specification of floral organidentity(see, e.g., Coen and Carpenter, supra). One central premise ofthis model is that AP2 and a second floral homeotic gene AGAMOUS (AG)are mutually antagonistic genes. That is, AP2 negatively regulates AGgene expression in sepals and petals, and conversely, AG negativelyregulates AP2 gene expression in stamens and carpels. In situhybridization analysis of AG gene expression in wild-type and ap2 mutantflowers has demonstrated that AP2 is indeed a negative regulator of AGexpression. However, it is not yet known how AP2 controls AG. Nor is itknown how AG influences AP2 gene activity.

The AP2 gene in Arabidopsis has been isolated by T-DNA insertionalmutagenesis as described in Jofuku et al. The Plant Cell 6:1211-1225(1994). AP2 encodes a putative nuclear factor that bears no significantsimilarity to any known fungal, or animal regulatory protein. Evidenceprovided there indicates that AP2 gene activity and function are notrestricted to developing flowers, suggesting that it may play a broaderrole in the regulation of Arabidopsis development than originallyproposed.

In spite of the recent progress in defining the genetic control of plantdevelopment, little progress has been reported in the identification andanalysis of genes effecting agronomically important traits such as seedsize, protein content, oil content and the like. Characterization ofsuch genes would allow for the genetic engineering of plants with avariety of desirable traits. The present invention addresses these andother needs.

SUMMARY OF THE INVENTION

The present invention provides methods of modulating seed mass and othertraits in plants. The methods involve providing a plant comprising arecombinant expression cassette containing an ADC nucleic acid linked toa plant promoter. The plant is either selfed or crossed with a secondplant to produce a plurality of seeds. Seeds with the desired trait(e.g., altered mass) are then selected.

In some embodiments, transcription of the ADC nucleic acid inhibitsexpression of an endogenous ADC gene or activity the encoded protein. Inthese embodiments, the step of selecting includes the step of selectingseed with increased mass or another trait. The seed may have, forinstance, increased protein content, carbohydrate content, or oilcontent. In the case of increased oil content, the types of fatty acidsmay or may not be altered as compared to the parental lines. In theseembodiments, the ADC nucleic acid may be linked to the plant promoter inthe sense or the antisense orientation. Alternatively, expression of theADC nucleic acid may enhance expression of an endogenous ADC gene or ADCactivity and the step of selecting includes the step of selecting seedwith decreased mass. This embodiment is particularly useful forproducing seedless varieties of crop plants.

If the first plant is crossed with a second plant the two plants may bethe same or different species. The plants may be any higher plants, forexample, members of the families Brassicaceae or Solanaceae. In makingseed of the invention, either the female or the male parent plant cancomprise the expression cassette containing the ADC nucleic acid. Inpreferred embodiments, both parents contain the expression cassette.

In the expression cassettes, the plant promoter may be a constitutivepromoter, for example, the CaMV 35S promoter. Alternatively, thepromoter may be a tissue-specific promoter. Examples of tissue specificexpression useful in the invention include fruit-specific, seed-specific(e.g., ovule-specific, embryo-specific, endosperm-specific,integument-specific, or seed coat-specifiic) expression.

The invention also provides seed produced by the methods describedabove. The seed of the invention comprise a recombinant expressioncassette containing an ADC nucleic acid. If the expression cassette isused to inhibit expression of endogenous ADC expression, the seed willhave a mass at least about 20% greater than the average mass of seeds ofthe same plant variety which lack the recombinant expression cassette.If the expression cassette is used to enhance expression of ADC, theseed will have a mass at least about 20% less than the average mass ofseeds of the same plant variety which lack the recombinant expressioncassette. Other traits such as protein content, carbohydrate content,and oil content can be altered in the same manner.

Definitions

The phrase "nucleic acid sequence" refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5'to the 3' end. It includes chromosomal DNA, self-replicating plasmids,infectious polymers of DNA or RNA and DNA or RNA that performs aprimarily structural role.

The term "promoter" refers to a region or sequence determinants locatedupstream or downstream from the start of transcription and which areinvolved in recognition and binding of RNA polymerase and other proteinsto initiate transcription. A "plant promoter" is a promoter capable ofinitiating transcription in plant cells.

The term "plant" includes whole plants, plant organs (e.g., leaves,stems, flowers, roots, etc.), seeds and plant cells and progeny of same.The class of plants which can be used in the method of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including angiosperms (monocotyledonous anddicotyledonous plants), as well as gymnosperms. It includes plants of avariety of ploidy levels, including polyploid, diploid, haploid andhemizygous.

A polynucleotide sequence is "heterologous to" an organism or a secondpolynucleotide sequence if it originates from a foreign species, or, iffrom the same species, is modified from its original form. For example,a promoter operably linked to a heterologous coding sequence refers to acoding sequence from a species different from that from which thepromoter was derived, or, if from the same species, a coding sequencewhich is different from any naturally occurring allelic variants. Asdefined here, a modified ADC coding sequence which is heterologous to anoperably linked ADC promoter does not include the T-DNA insertionalmutants (e.g, ap2-10) as described in Jofuku et al. The Plant Cell6:1211-1225 (1994).

A polynucleotide "exogenous to" an individual plant is a polynucleotidewhich is introduced into the plant by any means other than by a sexualcross. Examples of means by which this can be accomplished are describedbelow, and include Agrobacterium-mediated transformation, biolisticmethods, electroporation, and the like. Such a plant containing theexogenous nucleic acid is referred to here as an R₁ generationtransgenic plant. Transgenic plants which arise from sexual cross or byselfing are descendants of such a plant.

An "ADC (AP2 domain containing) nucleic acid" or "ADC polynucleotidesequence" of the invention is a subsequence or full lengthpolynucleotide sequence of a gene which, encodes an polypeptidecontaining an AP2 domain and when present in a transgenic plant, can beused to modulate seed properties in seed produced by the plant. Anexemplary nucleic acid of the invention is the Arabidopsis AP2 sequenceas disclosed in Jofuku et al. The Plant Cell 6:1211-1225 (1994). TheGenBank accession number for this sequence is U12546. As explained indetail below a family of RAP2 (related to AP2) genes have beenidentified in Arabidopsis. The class of nucleic acids claimed here fallsinto at least two subclasses (AP2-like and EREBP-like genes), which aredistinguished by, for instance, the number of AP2 domains containedwithin each polypeptide and by sequences within certain conservedregions. The differences between these two subclasses are described inmore detail below. ADC polynucleotides are defined by their ability tohybridize under defined conditions to the exemplified nucleic acids orPCR products derived from them. An ADC polynucleotide (e.g., AP2 orRAP2) is typically at least about 30-40 nucleotides to about 3000,usually less than about 5000 nucleotides in length. Usually the nucleicacids are from about 100 to about 2000 nucleotides, often from about 500to about 1700 nucleotides in length.

ADC nucleic acids, as explained in more detail below, are a new class ofplant regulatory genes that encode ADC polypeptides, which aredistinguished by the presence of one or more of a 56-68 amino acidrepeated motif, referred to here as the "AP2 domain". The amino acidsequence of an exemplary AP2 polypeptide is shown in Jofuku et al.,supra. One of skill will recognize that in light of the presentdisclosure various modifications (e.g., substitutions, additions, anddeletions) can be made to the sequences shown there withoutsubstantially affecting its function. These variations are specificallycovered by the terms ADC polypeptide or ADC polynucleotide.

In the case of both expression of transgenes and inhibition ofendogenous genes (e.g., by antisense, or sense suppression) one of skillwill recognize that the inserted polynucleotide sequence need not beidentical, but may be only "substantially identical" to a sequence ofthe gene from which it was derived. As explained below, thesesubstantially identical variants are specifically covered by the termADC nucleic acid.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the terms "ADC nucleic acid", "AP2 nucleic acid"and "RAP2 nucleic acid". In addition, the term specifically includesthose full length sequences substantially identical (determined asdescribed below) with an ADC polynucleotide sequence and that encodeproteins that retain the function of the ADC polypeptide (e.g.,resulting from conservative substitutions of amino acids in the AP2polypeptide). In addition, variants can be those that encode dominantnegative mutants as described below.

Two nucleic acid sequences or polypeptides are said to be "identical" ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term "complementary to" is used herein to mean thatthe complementary sequence is identical to all or a portion of areference polynucleotide sequence.

Sequence comparisons between two (or more) polynucleotides orpolypeptides are typically performed by comparing sequences of the twosequences over a "comparison window" to identify and compare localregions of sequence similarity. A "comparison window", as used herein,refers to a segment of at least about 20 contiguous positions, usuallyabout 50 to about 200, more usually about 100 to about 150 in which asequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

"Percentage of sequence identity" is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 60% sequenceidentity, preferably at least 80%, more preferably at least 90% and mostpreferably at least 95%, compared to a reference sequence using theprograms described above (preferably BLAST) using standard parameters.One of skill will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like. Substantial identityof amino acid sequences for these purposes normally means sequenceidentity of at least 35%, preferably at least 60%, more preferably atleast 90%, and most preferably at least 95 %. Polypeptides which are"substantially similar" share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast about 60°°C.

In the present invention, genomic DNA or cDNA comprising ADC nucleicacids of the invention can be identified in standard Southern blotsunder stringent conditions using the nucleic acid sequences disclosedhere. For the purposes of this disclosure, stringent conditions for suchhybridizations are those which include at least one wash in 0.2× SSC ata temperature of at least about 50° C., usually about 55° C. to about60° C., for 20 minutes, or equivalent conditions. Other means by whichnucleic acids of the invention can be identified are described in moredetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows amino acid sequence alignment between AP2 direct repeatsAP2-R1 (aa 129-195; SEQ ID NO:4) and AP2-R2 (aa 221-288; SEQ ID NO:5).Solid and dashed lines between the two sequences indicate residueidentity and similarity, respectively. Arrows indicate the positions ofthe ap2-1, ap2-5, and ap2-10 mutations described in Jofuku et al.(1994). The bracket above the AP2-R1 and AP2-R2 sequences indicates theresidues capable of forming amphipathic α-helices shown in FIG. 1B.

FIG. 1B is a schematic diagram of the putative AP2-R1 (R1; SEQ ID NO:6)and AP2-R2 (R2; SEQ ID NO:7) amphipathic α-helices. The NH2 terminalends of the R1 and R2 helices begin at residues Phe-160 and Phe-253 androtate clockwise by 100° per residue through Phe-177 and Cys-270,respectively. Arrows directed toward or away from the center of thehelical wheel diagrams indicate the negative or positive degree ofhydrophobicity as defined by ones et al. J. Lipid Res. 33: 87-296(1992).

FIG. 2 shows an antisense construct of the invention. pPW14.4 (which isidentical to pPW15) represents the 13.41 kb AP2 antisense gene constructused in plant transformation described here. pPW14.4 is comprised of theAP2 gene coding region in a transcriptional fusion with the cauliflowermosaic virus 35S (P35S) constitutive promoter in an antisenseorientation. The Ti plasmid vector used is a modified version of thepGSJ780A vector (Plant Genetic Systems, Gent, Belgium) in which a uniqueEcoR1 restriction site was introduced into the BamH1 site using aCla1-EcoR1-BamH1 adaptor. The modified pGSJ780A vector DNA waslinearized with EcoR1 and the AP2 coding region inserted as a 1.68 kbEcoR1 DNA fragment from AP2 cDNA plasmid cAP2#1 (Jofuku et al., 1994) inan antisense orientation with respect to the 35S promoter. KmRrepresents the plant selectable marker gene NPTII which confersresistance to the antibiotic kanamycin to transformed plant cellscarrying an integrated 35S-AP2 antisense gene. Boxes 1 and 5 representthe T-DNA left and right border sequences, respectively, that arerequired for transfer of T-DNA containing the 35S-AP2 antisense geneconstruct into the plant genome. Regions 2 and 3 contain T-DNAsequences. Box 3 designates the 3' octopine synthase gene sequences thatfunction in transcriptional termination. Region 6 designates bacterialDNA sequences that function as a bacterial origin of replication in bothE. coli and Agrobacterium tumefaciens, thus allowing pPW14.4 plasmidreplication and retention in both bacteria. Box 7 represents thebacterial selectable marker gene that confers resistance to theantibiotics streptomycin and spectinomycin and allows for selection ofAgrobacterium strains that carry the pPW14.4 recombinant plasmid.

FIG. 3 shows a sense construct of the invention. pPW12.4 (which isidentical to pPW9) represents the 13.41 kb AP2 sense gene construct usedin plant transformation described here. pPW12.4 is comprised of the AP2gene coding region in a transcriptional fusion with the cauliflowermosaic virus 35S (P35S) constitutive promoter in a sense orientation.The Ti plasmid vector used is a modified version of the pGSJ780A vector(Plant Genetic Systems, Gent, Belgium) in which a unique EcoR1restriction site was introduced into the BamH1 site using aCla1-EcoR1-BamH1 adaptor. The modified pGSJ780A vector DNA waslinearized with EcoR1 and the AP2 coding region inserted as a 1.68 kbEcoR1 DNA fragment from AP2 cDNA plasmid cAP2#1 (Jofuku et al., 1994) ina sense orientation with respect to the 35S promoter. KmR represents theplant selectable marker gene NPTII which confers resistance to theantibiotic kanamycin to transformed plant cells carrying an integrated35S-AP2 antisense gene. Boxes 1 and 5 represent the T-DNA left and rightborder sequences, respectively, that are required for transfer of T-DNAcontaining the 35S-AP2 sense gene construct into the plant genome.Regions 2 and 3 contain T-DNA sequences. Box 3 designates the 3'octopine synthase gene sequences that function in transcriptionaltermination. Region 6 designates bacterial DNA sequences that functionas a bacterial origin of replication in both E. coli and Agrobacteriumtumefaciens, thus allowing pPW12.4 plasmid replication and retention inboth bacteria. Box 7 represents the bacterial selectable marker genethat confers resistance to the antibiotics streptomycin andspectinomycin and allows for selection of Agrobacterium strains thatcarry the pPW12.4 recombinant plasmid.

FIGS. 4A and 4B show AP2 domain sequence and structure. The number ofamino acid residues within each AP2 domain is shown to the right.Sequence gaps were introduced to maximize sequence alignments. Theposition of amino acid residues and sequence gaps within the AP2 domainalignments are numbered 1-77 for reference. The location of theconserved YRG and RAYD (SEQ ID NO:8) elements are indicated by brackets.Shaded boxes highlight regions of sequence similarity. Positivelycharged amino acids within the YRG element are indicated by + signsabove the residues. The location of the 18-amino acid core region thatis predicted to form an amphipathic α-helix in AP2 is indicated by abracket. Residues within the RAYD (SEQ ID NO:8) element of each AP2domain that are predicted to form an amphipathic α-helix are underlined.FIG. 4A shows members of the AP2-like subclass. Amino acid sequencealignment between the AP2 domain repeats R1 and R2 contained within AP2,ANT and RAP2.7 AP2 (SEQ ID NOs:4 and 5), ANT (SEQ ID NOs:9 and 10) andRAP2.7 (SEQ ID NOs:11 and 12) is shown. Brackets above the sequencesdesignate the conserved YRG and RAYD (SEQ ID NO:8) blocks describedabove. The filled circle and asterisk indicate the positions of theap2-1, and ap2-5 mutations, respectively. Amino acid residues thatconstitute a consensus AP2 domain motif for AP2, ANT, and RAP2.7 isshown below the alignment with invariant residues shown capitalized (SEQID NOs:13-16). FIG. 4B shows members of the EREBP-like subclass. Aminoacid sequence alignment between the AP2 domains contained within thetobacco EREBPs (SEQ ID NOs:17-20) and the Arabidopsis EREBP-like RAP2proteins (SEQ ID NOs:21-31)is shown Consensus sequences are shown asabove (SEQ ID NOs:32-35). GenBank accession numbers for EREBP-1,EREBP-2, EREBP-3, and EREBP-4 are D38123, D38126, D38124, and D38125,respectively.

FIG. 4C provides schematic diagrams of the putative RAP2.7-R1(SEQ IDNO:36), AP2-R1 (SEQ ID NO:6), and ANT-R1 (SEQ ID NO:37) amphipathicα-helices. Amino acid residues within the RAP2.7-R1, AP2-R1, and ANT-R1motifs shown underlined in A that are predicted to form amphipathicα-helices are schematically displayed with residues rotating clockwiseby 100° per residue to form helical structures. Arrows directed towardor away from the center of the helical wheel diagrams indicate thenegative or positive degree of hydrophobicity as defined by Jones et al.J. Lipid Res. 33:287-296 (1992). Positively and negatively charged aminoacid residues are designated by + and - signs, respectively.

FIG. 4D shows schematic diagrams of the putative RAP2.2, RAP2.5,RAP2.12, and EREBP-3 amphipathic α-helices (SEQ ID NOs:38, 39,40 and41). Amino acid residues within the RAP2.2, RAP2.5, RAP2-12, and EREBP-3motifs shown underlined in FIG. 4B that are predicted to formamphipathic α-helices are schematically displayed as described in FIG.4C.

FIG. 4E shows sequence alignment between the 25-26 amino acid linkerregions in AP2, ANT, and RAP2.7 (SEQ ID NOs:42, 43 and 44, respectively.R1 and R2 designate the positions of the R1 and R2 repeats within AP2,ANT, and RAP2.7 relative to the linker region sequences. Boxes designateinvariant residues within the conserved linker regions. Amino acidresidues that constitute a consensus linker region motif for AP2, ANT,and RAP2.7 are shown below the alignment with invariant residues showncapitalized (SEQ ID NOs:45 and 46). The arrowhead indicates the positionof the ant-3 mutation described by Klucher et al. Plant Cell 8:137-153(1996).

FIG. 5 is a schematic diagram of pAP2, which can be used to constructexpression vectors of the invention.

FIG. 6 is a schematic diagram of pBEL1, which can be used to constructexpression vectors of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to plant ADC genes, such as the AP2 and RAP2genes of Arabidopsis. The invention provides molecular strategies forcontrolling seed size and total seed protein using ADC overexpressionand antisense gene constructs. In particular, transgenic plantscontaining antisense constructs have dramatically increased seed mass,seed protein, or seed oil. Alternatively, overexpression of ADC using aconstructs of the invention leads to reduced seed size and total seedprotein. Together, data presented here demonstrate that a number ofagronomically important traits including seed mass, total seed protein,and oil content, can be controlled in species of agriculturalimportance.

Isolation of ADC Nucleic Acids

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989).

The isolation of ADC nucleic acids may be accomplished by a number oftechniques. For instance, oligonucleotide probes based on the sequencesdisclosed here can be used to identify the desired gene in a cDNA orgenomic DNA library. To construct genomic libraries, large segments ofgenomic DNA are generated by random fragmentation, e.g. usingrestriction endonucleases, and are ligated with vector DNA to formconcatemers that can be packaged into the appropriate vector. To preparea cDNA library, mRNA is isolated from the desired organ, such asflowers, and a cDNA library which contains the ADC gene transcript isprepared from the mRNA. Alternatively, cDNA may be prepared from mRNAextracted from other tissues in which ADC genes or homologs areexpressed.

The cDNA or genomic library can then be screened using a probe basedupon the sequence of a cloned ADC gene disclosed here. Probes may beused to hybridize with genomic DNA or cDNA sequences to isolatehomologous genes in the same or different plant species. Alternatively,antibodies raised against an ADC polypeptide can be used to screen anmRNA expression library.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology can be used to amplify thesequences of the ADC genes directly from genomic DNA, from cDNA, fromgenomic libraries or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clone nucleicacid sequences that code for proteins to be expressed, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes.

Appropriate primers and probes for identifying ADC sequences from planttissues are generated from comparisons of the sequences provided inJofuku et al., supra. For a general overview of PCR see PCR Protocols: AGuide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J.and White, T., eds.), Academic Press, San Diego (1990).

As noted above, the nucleic acids of the invention are characterized bythe presence of sequence encoding a AP2 domain. Thus, these nucleicacids can be identified by their ability to specifically hybridize tosequences encoding AP2 domain disclosed here. Primers which specificallyamplify AP2 domains of the exemplified genes are particularly useful foridentification of particular ADC polynucleotides. Primers suitable forthis purpose based on the sequence of RAP2 genes disclosed here are asfollows:

    __________________________________________________________________________    SEQ ID NO: 47-60                                                                   GenBank                                                                             Name Number Primers                                                __________________________________________________________________________    AP2  U12546                                                                             JOAP2U 5'-GTTGCCGCTGCCGTAGTG-3'                                         JOAP2L 5'-GGTTCATCCTGAGCCGCATATC-3'                                         RAP2.1 AF003094 JORAP2.1U 5'-CTCAAGAAGAAGTGCCTAACCACG-3'                        JORAP2.1L 5'-GCAGAAGCTAGAAGAGCGTCGA-3'                                      RAP2.2 AF003095 JORAP2.2U 5'-GGAAAATGGGCTGCGGAG-3'                              JORAP2.2L 5'-GTTACCTCCAGCATCGAACGAG-3'                                      RAP2.4 AF003097 JORAP2.4U 5'-GCTGGATCTTGTTTCGCTTACG-3'                          JORAP2.4L 5'-GCTTCAAGCTTAGCGTCGACTG-3'                                      RAP2.5 AF003098 JORAP2.5U 5'-AGATGGGCTTGAAACCCGAC-3'                            JORAP2.5L 5'-CTGGCTAGGGCTACGCGC-3'                                          RAP2.6 AF003099 JORAP2.6U 5'-TTCTTTGCCTCCTCAACCATTG-3'                          JORAP2.6L 5'-TCTGAGTTCCAACATTTTCGGG-3'                                      RAP2.7 AF003100 JORAP2.7U 5'-GAAATTGGTAACTCCGGTTCCG-3'                          JORAP2.7L 5'-CCATTTTGCTTTGGCGCATTAC-3'                                      RAP2.8 AF003101 JORAP2.8U 5'-GGCGTTACGCCTCTACCGG-3'                             JORAP2.8L 5'-CGCCGTCTTCCAGAACGTTC-3'                                        RAP2.9 AF003102 JORAP2.9U 5'-ATCACGGATCTGGCTTGGTTC-3'                           JORAP2.9L 5'-GCCTTCTTCCGTATCAACGTCG-3'                                      RAP2.10 AF003103 JORAP2.10U 5'-GTCAACTCCGGCGGTTACG-3'                           JORAP2.10L 5'-TCTCCTTATATACGCCGCCGA-3'                                      RAP2.11 AF003104 JORAP2.11U 5'-GAGAAGAGCAAAGGCAACAAGAC-3'                       JORAP2.11L 5'-AGTTGTTAGGAAAATGGTTTGCG-3'                                    RAP2.12 AF003105 JORAP2.12U 5'-AAACCATTCGTTTTCACTTCGACTC-3'                     JORAP2.12L 5'-TCACAGAGCGTTTCTGAGAATTAGC-3'                                __________________________________________________________________________

The PCR primers are used under standard PCR conditions (described forinstance in Innis et al.) using the nucleic acids as identified in theabove GenBank accessions as a template. The PCR products generated byany of the reactions can then be used to identify nucleic acids of theinvention (e.g., from a cDNA library) by their ability to hybridize tothese products. Particularly preferred hybridization conditions use aHybridization Buffer consisting of: 0.25M Phosphate Buffer (pH 7.2), 1mM EDTA, 1% Bovine Serum Albumin, 7% SDS. Hybridizations then followedby a first wash with 2.0×SSC+0. 1% SDS or 0.39M Na+ and subsequentwashes with 0.2×SSC+0.1% SDS or 0.042M Na+. Hybridization temperaturewill be from about 45° C. to about 78° C., usually from about 50° C. toabout 70° C. Followed by washes at 18° C.

Particularly preferred hybridization conditions are as follows:

    ______________________________________                                        Hybridization Temp.                                                                       Hybrid. Time                                                                             Wash Buffer A                                                                            Wash Buffer B                               ______________________________________                                        78 degrees C.                                                                             48 hrs     18 degrees C.                                                                            18 degrees C.                                 70 degrees C. 48 hrs 18 degrees C. 18 degrees C.                              65 degrees C. 48 hrs 18 degrees C. 18 degrees C.                              60 degrees C. 72 hrs 18 degrees C. 18 degrees C.                              55 degrees C. 96 hrs 18 degrees C. 18 degrees C.                              45 degrees C. 200 hrs   18 degrees C. No wash                               ______________________________________                                    

If desired, primers that amplify regions more specific to particular ADCgenes can be used. The PCR products produced by these primers can beused in the hybridization conditions described above to isolate nucleicacids of the invention.

    __________________________________________________________________________    SEQ ID NO: 71-94                                                                    GenBank                                                                   Name Number Primers                                                         __________________________________________________________________________    AP2   U12546                                                                              AP2U 5'-ATGTGGGATCTAAACGACGCAC-3'                                     AP2L 5'-GATCTTGGTCCACGCCGAC-3'                                              RAP2.1 AF003094 RAP2.1U 5'-AAG AGG ACC ATC TCT CAG-3'                           RAP2.1L 5'-AAC ACT CGC TAG CTT CTC-3'                                       RAP2.2 AF003095 RAP2.2U 5'-TGG TTC AGC AGC CAA CAC-3'                           RAP2.2L 5'-CAA TGC ATA GAG CTT GAG G-3'                                     RAP2.4 AF003097 RAP2.4U 5'-ACG GAT TTC ACA TCG GAG-3'                           RAP2.4L 5'-CTA AGC TAG AAT CGA ATC C-3'                                     RAP2.5 AF003098 RAP2.5U 5'-TACCGGTTTCGCGCGTAG-3'                                RAP2.5L 5'-CACCTTCGAAATCAACGACCG-3'                                         RAP2.6 AF003099 RAP2.6U 5'-TTCCCCGAAAATGTTGGAACTC-3'                            RAP2.6L 5'-TGGGAGAGAAAAAATTGGTAGATCG-3'                                     RAP2.7 AF003100 RAP2.7U 5'-CGA TGG AGA CGA AGA CTC-3'                           RAP2.7L 5'-GTC GGA ACC GGA GTT ACC-3'                                       RAP2.8 AF003101 RAP2.8U 5'-TCA CTC AAA GGC CGA GAT C-3'                         RAP2.8L 5'-TAA CAA CAT CAC CGG CTC G-3'                                     RAP2.9 AF003102 RAP2.9U 5'-GTG AAG GCT TAG GAG GAG-3'                           RAP2.9L 5'-TGC CTC ATA TGA GTC AGA G-3'                                     RAP2.10 AF003103 RAP2.10U 5'-TCCCGGAGCTTTTAGCCG-3'                              RAP2.10L 5'-CAACCCGTTCCAACGATCC-3'                                          RAP2.11 AF003104 RAP2.11U 5'-TTCTTCACCAGAAGCAGAGCATG-3'                         RAP2.11L 5'-CTCCATTCATTGCATATAGGGACG-3'                                     RAP2.12 AF003105 RAP2.12U 5'-GCTTTGGTTCAGAACTCGAACATC-3'                        RAP2.12L 5'-AGGTTGATAAACGAACGAACGATGCG-3'                                 __________________________________________________________________________

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers et al.,Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams etal., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

Alternatively, primers that specifically hybridize to highly conservedregions in AP2 domains can be used to amplify sequences from widelydivergent plant species such as Arabidopsis, canola, soybean, tobacco,and snapdragon. Examples of such primers are as follows:

Primer RISZU 1 (SEQ ID NO:95): 5'-GGAYTGTGGGAAACAAGTTTA-3'

Primer RISZU 2 (SEQ ID NO:96): 5'-TGCAAAGTRACACCTCTATACTT-3'

Y=pyrimidine (T or C)

R=purine (A or G).

Standard nucleic acid hybridization techniques using the conditionsdisclosed above can then be used to identify full length cDNA or genomicclones.

In addition, the following DNA primers, RISZU 3 and RISZU 4, can be usedin an inverse PCR reaction to specifically amplify flanking AP2 genesequences from widely divergent plant species. These primers are asfollows:

Primer RISZU 3 (SEQ ID NO:97): 5'-GCATGWGCAGTGTCAAATCCA-3'

Primer RISZU 4 (SEQ ID NO:98): 5'-GAGGAAGTTCVAAGTATAGA-3'

W=A or T

V=G, A, or C

These primers have been used in standard PCR conditions to amplify ADCgene sequences from canola (SEQ ID NO:1) and soybean (SEQ ID NOS:2 and3).

Suppression of ADC Activity or Gene Expression

One of skill will recognize that a number of methods can be used toinactivate or suppress ADC activity or gene expression. The control ofthe expression can be achieved by introducing mutations into the gene orusing recombinant DNA techniques. These techniques are generally wellknown to one of skill and are discussed briefly below.

Methods for introducing genetic mutations into plant genes are wellknown. For instance, seeds or other plant material can be treated with amutagenic chemical substance, according to standard techniques. Suchchemical substances include, but are not limited to, the following:diethyl sulfate, ethylene imine, ethyl methanesulfonate andN-nitroso-N-ethylurea. Alternatively, ionizing radiation from sourcessuch as, for example, X-rays or gamma rays can be used. Desired mutantsare selected by assaying for increased seed mass, oil content and otherproperties.

Gene expression can be inactivated using recombinant DNA techniques bytransforming plant cells with constructs comprising transposons or T-DNAsequences. ADC mutants prepared by these methods are identifiedaccording to standard techniques. For instance, mutants can be detectedby PCR or by detecting the presence or absence of ADC mRNA, e.g., byNorthern blots. Mutants can also be selected by assaying for increasedseed mass, oil content and other properties.

The isolated sequences prepared as described herein, can also be used ina number of techniques to suppress endogenous ADC gene expression. Aparticularly useful genes for this purpose are the AP2 gene described inJofuku et al., supra, and RAP2 genes described below. For instance,sequences flanking the AP2 domains of the genes disclosed here tospecifically target individual genes.

A number of methods can be used to inhibit gene expression in plants.For instance, antisense technology can be conveniently used. Toaccomplish this, a nucleic acid segment from the desired gene is clonedand operably linked to a promoter such that the antisense strand of RNAwill be transcribed. The construct is then transformed into plants andthe antisense strand of RNA is produced. In plant cells, it has beensuggested that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiattet al., U.S. Pat. No. 4,801,340.

The nucleic acid segment to be introduced generally will besubstantially identical to at least a portion of the endogenous ADC geneor genes to be repressed. The sequence, however, need not be perfectlyidentical to inhibit expression. The vectors of the present inventioncan be designed such that the inhibitory effect applies to other geneswithin a family of genes exhibiting homology or substantial homology tothe target gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about full length nucleotides should be used,though a sequence of at least about 100 nucleotides is preferred, asequence of at least about 200 nucleotides is more preferred, and asequence of about 500 to about 1700 nucleotides is especially preferred.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of ADC genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs which arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense cosuppression. Introduction ofnucleic acid configured in the sense orientation has been recently shownto be an effective means by which to block the transcription of targetgenes. For an example of the use of this method to modulate expressionof endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990),and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

The suppressive effect may occur where the introduced sequence containsno coding sequence per se, but only intron or untranslated sequenceshomologous to sequences present in the primary transcript of theendogenous sequence. The introduced sequence generally will besubstantially identical to the endogenous sequence intended to berepressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For sense suppression, the introduced sequence, needing less thanabsolute identity, also need not be full length, relative to either theprimary transcription product or fully processed mRNA. This may bepreferred to avoid concurrent production of some plants which areoverexpressers. A higher identity in a shorter than full length sequencecompensates for a longer, less identical sequence. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andidentity of non-coding segments will be equally effective. Normally, asequence of the size ranges noted above for antisense regulation isused.

Some ADC proteins (e.g., AP2) are believed to form multimers in vivo. Asa result, an alternative method for inhibiting ADC function is throughuse of dominant negative mutants. This approach involves transformationof plants with constructs encoding mutant ADC polypeptides that formdefective multimers with endogenous wild-type ADC proteins and therebyinactivate the protein. The mutant polypeptide may vary from thenaturally occurring sequence at the primary structure level by aminoacid substitutions, additions, deletions, and the like. Thesemodifications can be used in a number of combinations to produce thefinal modified protein chain. Use of dominant negative mutants toinactivate AG is described in Mizukami et al. Plant Cell 8:831-845(1996).

Use of Nucleic Acids of the Invention to Enhance ADC Gene Expression

Isolated sequences prepared as described herein can also be used tointroduce expression of a particular ADC nucleic acid to enhance orincrease endogenous gene expression. Enhanced expression will generallylead to smaller seeds or seedless fruit. Where overexpression of a geneis desired, the desired gene from a different species may be used todecrease potential sense suppression effects.

One of skill will recognize that the polypeptides encoded by the genesof the invention, like other proteins, have different domains whichperform different functions. Thus, the gene sequences need not be fulllength, so long as the desired functional domain of the protein isexpressed. The distinguishing features of ADC polypeptides, includingthe AP2 domain, are discussed in detail below.

Modified protein chains can also be readily designed utilizing variousrecombinant DNA techniques well known to those skilled in the art anddescribed in detail, below. For example, the chains can vary from thenaturally occurring sequence at the primary structure level by aminoacid substitutions, additions, deletions, and the like. Thesemodifications can be used in a number of combinations to produce thefinal modified protein chain.

Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of plant cells are prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988). ADNA sequence coding for the desired polypeptide, for example a cDNAsequence encoding a full length protein, will preferably be combinedwith transcriptional and translational initiation regulatory sequenceswhich will direct the transcription of the sequence from the gene in theintended tissues of the transformed plant.

For example, for overexpression, a plant promoter fragment may beemployed which will direct expression of the gene in all tissues of aregenerated plant. Such promoters are referred to herein as"constitutive" promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1'- or 2'- promoter derivedfrom T-DNA of Agrobacterium tumafaciens, and other transcriptioninitiation regions from various plant genes known to those of skill.Such genes include for example, the AP2 gene, ACT11 from Arabidopsis(Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis(GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)),the gene encoding stearoyl-acyl carrier protein desaturase from Brassicanapus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176(1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol.Biol 208:551-565 (1989)), and Gpc2 from maize (GenBank No. U45855,Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)).

Alternatively, the plant promoter may direct expression of the ADCnucleic acid in a specific tissue or may be otherwise under more preciseenvironmental or developmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, or the presence of light.Such promoters are referred to here as "inducible" or "tissue-specific"promoters. One of skill will recognize that a tissue-specific promotermay drive expression of operably linked sequences in tissues other thanthe target tissue. Thus, as used herein a tissue-specific promoter isone that drives expression preferentially in the target tissue, but mayalso lead to some expression in other tissues as well.

Examples of promoters under developmental control include promoters thatinitiate transcription only (or primarily only) in certain tissues, suchas fruit, seeds, or flowers. Promoters that direct expression of nucleicacids in ovules, flowers or seeds are particularly useful in the presentinvention. As used herein a seed-specific promoter is one which directsexpression in seed tissues, such promoters may be, for example,ovule-specific, embryo-specific, endosperm-specific,integument-specific, seed coat-specific, or some combination thereof.Examples include a promoter from the ovule-specific BEL1 gene describedin Reiser et al. Cell 83:735-742 (1995) (GenBank No. U39944). Othersuitable seed specific promoters are derived from the following genes:MAC1 from maize (Sheridan et al. Genetics 142:1009-1020 (1996), Cat3from maize (GenBank No. L05934, Abler et al. Plant Mol. Biol.22:10131-1038 (1993), the gene encoding oleosin 18 kD from maize(GenBank No. J05212, Lee et al. Plant Mol. Biol. 26:1981-1987 (1994)),vivparous-1 from Arabidopsis (Genbank No. U93215), the gene encodingoleosin from Arabidopsis (Genbank No. Z17657), Atmyc1 from Arabidopsis(Urao et al. Plant Mol. Biol. 32:571-576 (1996), the 2s seed storageprotein gene family from Arabidopsis (Conceicao et al. Plant 5:493-505(1994)) the gene encoding oleosin 20 kD from Brassica napus (GenBank No.M63985), napA from Brassica napus (GenBank No. J02798, Josefsson et al.JBL 26:12196-1301 (1987), the napin gene family from Brassica napus(Sjodahl et al. Planta 197:264-271 (1995), the gene encoding the 2Sstorage protein from Brassica napus (Dasgupta et al. Gene 133:301-302(1993)), the genes encoding oleosin A (Genbank No. U09118) and oleosin B(Genbank No. U09119) from soybean and the gene encoding low molecularweight sulphur rich protein from soybean (Choi et al. Mol Gen, Genet.246:266-268 (1995)).

If proper polypeptide expression is desired, a polyadenylation region atthe 3'-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes of the invention will typically comprise a marker gene whichconfers a selectable phenotype on plant cells. For example, the markermay encode biocide resistance, particularly antibiotic resistance, suchas resistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or Basta.

Production of Transgenic Plants

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal. Embo J. 3:2717-2722 (1984). Electroporation techniques are describedin Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistictransformation techniques are described in Klein et al. Nature 327:70-73(1987).

Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. Agrobacterium tumefaciens-mediated transformation techniques,including disarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch et al. Science233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803(1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotypesuch as increased seed mass. Such regeneration techniques rely onmanipulation of certain phytohormones in a tissue culture growth medium,typically relying on a biocide and/or herbicide marker which has beenintroduced together with the desired nucleotide sequences. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp.124-176, MacMillilan Publishing Company, New York, 1983; and Binding,Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, BocaRaton, 1985. Regeneration can also be obtained from plant callus,explants, organs, or parts thereof. Such regeneration techniques aredescribed generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486(1987).

The nucleic acids of the invention can be used to confer desired traitson essentially any plant. Thus, the invention has use over a broad rangeof plants, including species from the genera Anacardium, Arachis,Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus,Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana,Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea,Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum,Vicia, Vitis, Vigna, and Zea.

Increasing seed size, protein, amino acid, and oils content isparticularly desirable in crop plants in which seed are used directlyfor animal or human consumption or for industrial purposes. Examplesinclude soybean, canola, and grains such as rice, wheat, corn, rye, andthe like. Decreasing seed size, or producing seedless varieties, isparticularly important in plants grown for their fruit and in whichlarge seeds may be undesirable. Examples include cucumbers, tomatoes,melons, and cherries.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

Since transgenic expression of the nucleic acids of the invention leadsto phenotypic changes in seeds and fruit, plants comprising theexpression cassettes discussed above must be sexually crossed with asecond plant to obtain the final product. The seed of the invention canbe derived from a cross between two transgenic plants of the invention,or a cross between a plant of the invention and another plant. Thedesired effects (e.g., increased seed mass) are generally enhanced whenboth parental plants contain expression cassettes of the invention.

Seed obtained from plants of the present invention can be analyzedaccording to well known procedures to identify seed with the desiredtrait. Increased or decreased size can be determined by weighing seedsor by visual inspection. Protein content is conveniently measured by themethod of Bradford et al. Anal. Bioch. 72:248 (1976). Oil content isdetermined using standard procedures such as gas chromatography. Theseprocedures can also be used to determine whether the types of fattyacids and other lipids are altered in the plants of the invention.

Using these procedures one of skill can identify the seed of theinvention by the presence of the expression cassettes of the inventionand increased seed mass. Usually, the seed mass will be at least about10%, often about 20% greater than the average seed mass of plants of thesame variety that lack the expression cassette. The mass can be about50% greater and preferably at least about 75% to about 100% greater.Increases in other properties e.g., protein and oil will usually beproportional to the increases in mass. Thus, in some embodiments proteinor oil content can increase by about 10%, 20%, 50%, 75% or 100%, or inapproximate proportion to the increase in mass.

Alternatively, seed of the invention in which AP2 expression is enhancedwill have the expression cassettes of the invention and decreased seedmass. Seed mass will be at least about 20% less than the average seedmass of plants of the same variety that lack the expression cassette.Often the mass will be about 50% less and preferably at least about 75 %less or the seed will be absent. As above, decreases in other propertiese.g., protein and oil will be proportional to the decreases in mass.

The following Examples are offered by way of illustration, notlimitation.

EXAMPLE 1 AP2 Gene Isolation

The isolation and characterization of an AP2 gene from Arabidopsis isdescribed in detail in Jofuku et al., supra. Briefly, T-DNA fromAgrobacterium was used as an insertional mutagen to identify and isolategenes controlling flower formation in Arabidopsis. One transformed line,designated T10, segregated 3 to 1 for a flower mutant thatphenotypically resembled many allelic forms of the floral homeoticmutant ap2. T10 was tested and it was confirmed genetically that T10 andap2 are allelic. The mutant was designated as ap2-10.

It was determined that ap2-10 was the product of a T-DNA insertionmutation by genetic linkage analysis using the T-DNA-encoded neomycinphosphotransferase II (NPTII) gene as a genetic marker. An overlappingset of T-DNA-containing recombinant phage was selected from an ap2-10genome library and the plant DNA sequences flanking the T-DNA insertionelement were used as hybridization probes to isolate phage containingthe corresponding region from a wild-type Arabidopsis genome library.The site of T-DNA insertion in ap2-10 was mapped to a 7.2-kb EcoR1fragment centrally located within the AP2 gene region.

Five Arabidopsis flower cDNA clones corresponding to sequences withinthe 7.2-kb AP2 gene region were isolated. All five cloned cDNAs wereconfirmed to represent AP2 gene transcripts using an antisense genestrategy to induce ap2 mutant flowers in wild-type plants.

To determine AP2 gene structure, the nucleotide sequences of the cDNAinserts were compared to that of the 7.2-kb AP2 genomic fragment. Theseresults showed that the AP2 gene is 2.5 kb in length and contains 10exons and 9 introns that range from 85 to 110 bp in length. The AP2 geneencodes a theoretical polypeptide of 432 amino acids with a predictedmolecular mass of 48 kD. The AP2 nucleotide and predicted proteinsequences were compared with a merged, nonredundant data base. It wasfound that AP2 had no significant global similarity to any knownregulatory protein.

Sequence analysis, however, did reveal the presence of several sequencefeatures that may be important for AP2 protein structure or function.First, AP2 contains a 37-amino acid serine-rich acidic domain (aminoacids 14 to 50) that is analogous to regions that function as activationdomains in a number of RNA polymerase II transcription factors. Second,AP2 has a highly basic 10-amino acid domain (amino acids 119 to 128)that includes a putative nuclear localization sequence KKSR (SEQ IDNO:99) suggesting that AP2 may function in the nucleus. Finally, thatthe central core of the AP2 polypeptide (amino acids 129 to 288)contains two copies of a 68-amino acid direct repeat that is referred tohere as the AP2 domain. The two copies of this repeat, designated AP2-R1and AP2-R2, share 53% amino acid identity and 69% amino acid homology.FIG. 1A shows that each AP2 repeat contains an 18-amino acid conservedcore region that shares 83% amino acid homology. FIG. 1B shows that bothcopies of this core region are theoretically capable of formingamphipathic α-helical structures that may participate in protein-proteininteractions.

EXAMPLE 2 Preparation of AP2 Constructs

Gene constructs were made comprising the AP2 gene coding regiondescribed above in a transcriptional fusion with the cauliflower mosaicvirus 35S constitutive promoter in both the sense and antisenseorientations. The original vector containing the 35S promoter pGSJ780Awas obtained from Plant Genetic Systems (Gent, Belgium). The pGSJ780Avector was modified by inserting a Cla1-BamH1 adaptor containing anEcoR1 site in the unique BamH 1 site of pGSJ780A. The modified pGSJ780ADNA was linearized with EcoR1 and the AP2 gene coding region inserted asa 1.68 kb EcoR1 fragment in both sense and antisense orientations withrespect to the 35S promoter (see, FIGS. 2 and 3).

The resultant DNA was transformed into E. coli and spectinomycinresistant transformants were selected. Plasmid DNAs were isolated fromindividual transformants and the orientation of the insert DNAs relativeto the 35S promoter were confirmed by DNA sequencing. Bacterial cellscontaining the 35S/AP2 sense (designated pPW12.4 and pPW9) and 35S/AP2antisense (designated pPW14.4 and pPW15) constructs were conjugated toAgrobacterium tumefaciens and rifampicin, spectinomycin resistanttransformants were selected for use in Agrobacterium-mediated planttransformation experiments.

The 35S/AP2 sense and 35S/AP2 antisense constructs were introduced intowild-type Arabidopsis and tobacco plants according to standardtechniques. Stable transgenic plant lines were selected using the plantselectable marker NPTII (which confers resistance to the antibiotickanamycin) present on the modified Ti plasmid vector pGSJ780A.

EXAMPLE 3 Modification of Seed using AP2 Sequences

This example shows that ap2 mutant plants and transgenic plantscontaining the 35S/AP2 antisense construct produced seed with increasedmass and total protein content. By contrast, transgenic plantscontaining the 35S/AP2 sense construct produced seed with decreased massand protein content. Together these results indicate that seed mass andseed contents in transgenic plants can be modified by geneticallyaltering AP2 activity.

Seed from 30 lines were analyzed for altered seed size and seed proteincontent including the Arabidopsis ap2 mutants ap2-1, ap2-3, ap2-4,ap2-5, ap2-6, ap2-9 and ap2-10 and transgenic Arabidopsis and transgenictobacco containing the CaMV 35S/AP2 antisense gene construct, the CaMV35S/AP2 sense gene construct, or the pGSJ780A vector as described above.The ap2 mutants used in this study are described in Komaki et al.,Development 104, 195-203 (1988), Kunst et al., Plant Cell 1, 1195-1208(1989), Bowman et al., Development 112, 1-20 (1991), and Jofuku et al.,supra.

Due to the small size of Arabidopsis and tobacco seed, average seed masswas determined by weighing seed in batches of 100 for Arabidopsis and 50seed for tobacco. The net change in seed mass due to changes in AP2 geneactivity was calculated by subtracting the average mass of wild-typeseed from mutant seed mass.

Seed from three wild-type Arabidopsis ecotypes C24, Landsberg-er ,andColumbia, and one wild-type tobacco SR1 were used as controls. Wild-typeArabidopsis seed display seasonal variations in seed mass which rangefrom 1.6-2.3 mg per 100 seed as shown in Table I. Therefore transgenicArabidopsis seed were compared to control seed that had been harvestedat approximately the same time of season. This proved to be an importantfor comparing the effects of weak ap2 mutations on seed mass.

Table I shows that all ap2 mutant seed examined, ap2-1, ap2-3, ap2-4,ap2-5, ap2-6, ap2-9, and ap2-10, show a significant increase in averageseed mass ranging from +27 to +104 percent compared to wild-type. Theweak partial loss-of-function mutants such as ap2-1 and ap2-3 show thesmallest gain in average seed mass ranging from +27 percent to +40percent of wild-type, respectively. By contrast, strong ap2 mutants suchas ap2-6 and ap2-10 show the largest gain in seed mass ranging from +69percent to +104 percent of wild-type, respectively. Thus reducing AP2gene activity genetically consistently increases Arabidopsis seed mass.

AP2 antisense and AP2 sense cosuppression strategies described abovewere used to reduce AP2 gene activity in planta to determine whetherseed mass could be manipulated in transgenic wild-type plants.Twenty-nine independent lines of transgenic Arabidopsis containing theCaMV 35S/AP2 antisense gene constructs pPW14.4 and pPW15 (FIG. 2) weregenerated. Each transgenic line used in this study tested positive forkanamycin resistance and the presence of one or more copies of T-DNA.

Table I shows that seed from nine transgenic Arabidopsis AP2 antisenselines show a significant increase in seed mass when compared to controlseed ranging from +22 percent for line C24 15-542 to +89 percent forline C24 15-566. Both C24 and Landsberg-er ecotypes were usedsuccessfully. Increased seed mass was observed in F1, F2, and F3generation seed.

Eight lines containing the 35S/AP2 sense gene construct were generatedwhich were phenotypically cosuppression mutants. As shown in Table Iseed from two cosuppression lines examined showed larger seed that rangefrom +26 percent to +86 percent. By contrast, plants transformed withthe vector pGSJ780A showed a normal range of average seed mass rangingfrom -0.5 percent to +13 percent compared to wild-type seed (Table I).Together, these results demonstrate that AP2 gene sequences can be usedto produce a significant increase in Arabidopsis seed mass using bothantisense and cosuppression strategies in a flowering plant.

                  TABLE I                                                         ______________________________________                                        Genetic control of Arabidopsis seed mass by AP2                                                            Percent change                                     Average seed in seed mass                                                     mass in mg per compared to                                                    100 seed.sup.1,2 wild-type                                                  ______________________________________                                        ap2 mutant seed  2.1 (0.1)   +27%                                             1.  ap2-1            2.2 (0.1)   +33%                                             2.1 (0.2) +31%                                                                2.8 (0.2) +33%                                                              2. ap2-3 2.6 (0.1) +27%                                                       3. ap2-4 3.5 (0.3) +69%                                                         3.5 (0.2) +69%                                                              4. ap2-5 2.9 (0.1) +39%                                                       5. ap2-6 3.5 (0.2) +69%                                                       6. ap2-9 2.9 (0.1) +40%                                                       7. ap2-10 3.7 (0.4) +79%                                                        3.9 (0.3) +90%                                                                4.2 (0.5) +104%                                                           Seed produced by transgenic CaMV35S/AP2 antisense lines                         (from a Km resistant mother)                                                  1.    C24 14.4E (F1-15) F2 sd                                                                        3.1  .sup.                                                                              +35%                                          C24 14.4E (F1-15) F3 sd 3.4 (0.3) +47%                                       2. C24 14.4S (F1-1) 2.8 (0.2) +29%                                            3. C24 14.4AA (F1-24) 2.9 (0.1) +30%                                          4. C24 14.4DD (F1-2) 2.8 (0.3) +30%                                           5. C24 15-522 3.6 (0.1) +76%                                                  6. C24 15-542 (F1-2) 2.6 (0.1) +25%                                            C24 15-542 (F1-7) 2.5 (0.2) +22%                                             7. C24 15-566 3.9 (0.1) +89%                                                  8. LE 15-9992-3 (F1-1) F2 sd 2.4 (0.1) +42%                                   9. LE 15-83192-3 (F1-3) 2.8 (0.0) +33%                                         LE 15-83192-3 (F1-17) 2.7 (0.0) +28%                                       Seed produced by transgenic CaMV35S/AP2 cosuppression lines                     (from a Km resistant mother)                                                  1.    C24 9-5 (F1-5)   3.8 (0.0) +86%                                         2. LE 9-83192--2 (F1-19) 2.7 (0.2) +26%                                        LE 9-83192-2 (F1-24) 2.7 (0.1) +26%                                        Seed produced by transgenic pGSJ780A vector only lines                          (from a Km resistant mother plant)                                            1.    C24 3-107 (F1-1) 2.2 (0.1)  +9%                                         2. C24 3-109 (F1-1) 2.3 (0.0) +13%                                            3. LE 3-83192-1 (F1-2) 2.3 (0.1)  +7%                                         4. LE 3-83192-3 (F1-2) 2.4 (0.1) +11%                                         5. LE 3-9992-4 (F1-4) 2.4 (0.2) +12%                                           LE 3-9992-4 (F1-6) 2.3 (0.0)  +9%                                             LE 3-9992-4 (F1-8) 2.1 (0.0)   -0.5%                                         6. LE 3-9992-9 (F1-3) 2.3 (0.1)  +7%                                        Seed produced by wild-type Arabidopsis plants                                   1.    C24              2.0 (0.1)                                                2.3 (0.1)                                                                     2.2                                                                         2. Landsberg-er 1.6 (0.1)                                                       2.1 (0.1)                                                                     2.1                                                                           2.3 (0.1)                                                                   3. Columbia 1.8 (0.1)                                                           2.1 (0.1)                                                                 ______________________________________                                         .sup.1 Standard deviation values are given in parentheses.                    .sup.2 Wildtype seed values used for this comparison were chosen by           ecotype and harvest date.                                                

Arabidopsis AP2 gene sequences were also used to negatively control seedmass in tobacco, a heterologous plant species. Table II shows that infive transgenic tobacco lines the CAMV 35S/AP2 overexpression geneconstruct was effective in reducing transgenic seed mass from -27percent to -38 percent compared to wild-type seed. These resultsdemonstrate the evolutionary conservation of AP2 gene function at theprotein level for controlling seed mass in a heterologous system.

                  TABLE II                                                        ______________________________________                                        Genetic control of tobacco seed mass using Arabidopsis AP2                                 Average seed                                                       mass in mg Percent change in seed mass                                        per 5 seed.sup.1 compared to wild-type                                      ______________________________________                                        Seed produced by transgenic CaMV 35S/AP2 sense gene lines                       (from a Km resistant mother)                                                  1.    SR1 9-110 To 3.1 (0.0)                                                                              -27%                                               SR1 9-110 (F1-5) 3.0 (0.2) -29%                                                2.8 (0.3) -34%                                                              2. SR1 9-202 (F1-G) 3.1 (0.2) -27%                                             SR1 9-202 (F1-I) 3.2 (0.1) -24%                                              3. SR1 9-103 (F1-2) 3.9 (0.0)  -8%                                            4. SR1 9-413-1 2.8 (0.0) -34%                                                   3.0 (0.2) -29%                                                              5. SR1 9-418-1 To 3.5 (0.1) -18%                                            Seed produced by transgenic CaMV 35S/AP2 antisense gene lines                   (from a Km resistant mother)                                                  1.    SR1 15-111   5.1 (0.4)                                                                              +20%                                               SR1 15-111 (F1) 5.0 (0.4) +19%                                               2. SR1 15-116 To 4.1 (0.4)  -3%                                                SR1 15-116 (F1-2) 4.0 (0.1)  -5%                                              SR1 15-116 (F1-1) 4.5 (0.1)  +5%                                             3. SR1 15-407 (F1) 4.8 (0.5) +10%                                               4.7 (0.3) +10%                                                              4. SR1 15-102 (F1) 4.5 (0.2)  +6%                                             5. SR1 15-413 (F1-3) 4.2 (0.0)  +0%                                           6. SR1 15-410 (F1-2) 4.4 (0.0)  +4%                                           7. SR1 15-210 (F1-4) 3.6 (0.1) -15%                                         Seed produced by pGSJ780A vector only lines                                     (from a Km resistant mother)                                                  1.    SR1 3-402 (F1)                                                                             5.0 (0.1)                                                                              +17%                                              2. SR1 3-401 (F1) 4.6 (0.1)  +8%                                              3. SR1 3-405 (F1) 4.4 (0.1)  +4%                                            Seed from wild-type tobacco                                                     1.    SR1          4.2 (0.3)                                                    4.0 (0.1)                                                                 ______________________________________                                         .sup.1 Standard deviation values are given in parentheses.               

Use of AP2 Gene Constructs to Control Seed Protein Content

Total seed protein was extracted and quantitated from seed produced bywild-type, ap2 mutant, transgenic AP2 antisense, and transgenic AP2sense cosuppression plants according to Naito et al. Plant Mol Biol. 11,109-123 (1988). Seed protein was extracted in triplicate from batches of100 dried seed for Arabidopsis or 50 dried seed for tobacco. Totalprotein yield was determined by the Bradford dye-binding procedure asdescribed by Bradford, Anal. Biochem. 72:248 (1976). The results of thisanalysis are shown in Table III.

ap2 mutant total seed protein content increased by 20 percent to 78percent compared to wild-type control seed. Total seed protein fromtransgenic AP2 antisense plants increased by +31 percent to +97 percentcompared to wild-type controls. Transgenic AP2 cosuppression seed showeda +13 and +17 percent increase over wild-type. Together, the transgenicantisense and cosuppression mutant seed consistently yielded moreprotein per seed than did the wild-type controls or transgenic plantscontaining the pGSJ780A vector only (Table III).

                  TABLE III                                                       ______________________________________                                        Genetic control of total seed protein content in Arabidopsis using AP2                       Total see protein                                                                         Percent change in                                    in μg per protein content                                                  100 seed.sup.1 compared to wild-type                                        ______________________________________                                        ap2 mutant seed                                                                 1.    ap2-1          652 (17)  +20%                                              relative to WT seed                                                          615 (30) +11%                                                               2. ap2-3 705 (47) +27%                                                        3. ap2-4 729 (107) +33%                                                       4. ap2-5 617 (24) +13%                                                        5. ap2-6 836 (14) +52%                                                        6. ap2-9 798 (11) +46%                                                        7. ap2-10 836 (15) +78%                                                     Transgenic CaMV 35S/AP2 antisense see mass                                      (from Km resistant mother)                                                    1.    C24 14.4E (F1-1) F3 sd                                                                       615 (60)  +31%                                           2. C24 15-522 (F1-1) 790 (23) +68%                                            3. C24 15-566 925 (173) +97%                                                Transgenic CaMV 35S/AP2 sense cosuppression seed mass                           (from Km Resistant mother plant)                                              1.    LE9-83192-2 (F1-19)                                                                          616       +13%                                            LE9-83192-2 (F1-24) 637 +17%                                               Wild-type seed                                                                  1.    C24            469 (19)                                                 2. LE 545 (22)                                                                  555                                                                         3. Col 548 (42)                                                             ______________________________________                                         .sup.1 Standard deviation values are given in parentheses.               

Transgenic tobacco containing the 35S/AP2 sense gene construct show thatAP2 overexpression can decrease seed protein content by 27 to 45 percentcompared to wild-type seed. Together, the transgenic Arabidopsis andtobacco results demonstrate that seed mass and seed protein productioncan be controlled by regulating AP2 gene activity.

                  TABLE IV                                                        ______________________________________                                        Negative control of transgenic tobacco seed protein content                     by Arabidopsis AP2 gene expression.sup.1                                                             Percent change in protein                              Ave. protein content compared to                                              per 50 seed wild-type                                                       ______________________________________                                        Seed produced by transgenic CAMV 35S/AP2 sense gene plant                       1.     SR1 9-110    242 (11) -45%                                             2. SR1 9-202 (F1-G) 271 (11) -38%                                             3. SR1 9-413 362 (8)  -18%                                                    4. SR1 9-418-1 319 (16) -27%                                                Wild-type Control                                                               SR1 (wild-type) 440 (8) (JO)                                                                             NA                                               ______________________________________                                         .sup.1 Standard deviation values are given in parentheses.               

Analysis of Transgenic Seed Proteins by Gel Electrophoresis

Arabidopsis seed produce two major classes of seed storage proteins, the12S cruciferins and 2S napins which are structurally related to themajor storage proteins found in the Brassicaceae and in theLeguminoceae. The composition of seed proteins in wild-type, ap2mutant,and transgenic Arabidopsis seed were compared by SDSpolyacrylamide gel electrophoresis as described by Naito et al., PlantMol. Biol. 11, 109-123 (1988). Total seed proteins were extracted asdescribed above. 50 μg aliquots were separated by gel electrophoresisand stained using Coomassie brilliant blue. These results showed thatthe spectrum of proteins in wild-type and ap2 mutant seed arequalitatively indistinguishable. There is no detectable difference inthe representation of the 12S or 2S storage proteins between thewild-type and ap2 mutant seed extracts. This shows that reducing AP2gene activity genetically does not alter the profile of storage proteinssynthesized during seed maturation. The spectrum of seed proteinsproduced in transgenic AP2 antisense and AP2 sense cosuppression seedare also indistinguishable from wild-type. In particular, there is nodetectable difference in the representation of the 12S cruciferin or 2Snapin storage proteins in the larger seed.

Finally, the transgenic tobacco plants containing the 35S/AP2overexpression gene construct produced significantly smaller seed.Despite the decrease in seed mass in transgenic tobacco there was nodetectable difference in storage protein profiles between seed from35S/AP2 transformants and wild-type SR1.

EXAMPLE 4 Isolation of Other Members of the AP2 Gene Family fromArabidopsis

This example describes isolation of a number of AP2 nucleic acids fromArabidopsis. The nucleic acids are referred to here as RAP2 (related toAP2) were identified using primers specific to nucleic acid sequencesfrom the AP2 domain described above.

MATERIALS AND METHODS

Plant Material. Arabidopsis thaliana ecotype Landsberg erecta (L-er) andC24 were used as wild type. Plants were grown at 22° C. under a 16-hrlight/8-hr dark photoperiod in a 1:1:1 mixture containingvermiculite/perlite/peat moss. Plants were watered with a one-fourthstrength Peter's solution (Grace-Sierra, Milpitas, Calif.). Root tissuewas harvested from plants grown hydroponically in sterile flaskscontaining 1× Murashige and Skoog plant salts (GIBCO), 1 mg/literthiamine, 0.5 mg/liter pyridoxine, 0.5 mg/liter nicotinic acid, 0.5g/liter 2-(N-morpholino)ethanesulfonic acid (MES), and 3 % sucrose, withmoderate shaking and 70 μmol -m⁻² -sec⁻¹ of light.

Analysis of cloned Arabidopsis cDNAs. Arabidopsis expressed sequencetagged (EST) CDNA clones representing RAP2.1 and RAP2.9 were generatedas described by Cooke et al. (Cooke, R., et al., 1996, Plant J. 9,101-124). EST cDNA clones representing RAP2.2 and RAP2.8 were generatedas described by Hofte et al. (Hofte, H., et al., 1993, Plant J. 4,1051-1061). EST cDNA clones representing all other RAP2 genes weregenerated by Newman et al. (Newman, T., et al., 1994, Plant Physiol.106,1241-1255) and provided by the Arabidopsis Biological ResourceCenter (Ohio State University). Plasmid DNAs were isolated and purifiedby anion exchange chromatography (Qiagen, Chatsworth, Calif.). DNAsequences were generated using fluorescence dye-based nucleotideterminators and analyzed as specified by the manufacturer (AppliedBiosystems).

Nucleotide and Amino Acid Sequence Comparisons. The TBLASTN program(Altschul, S. F., et al., 1990, J. Mol. Biol. 215, 403-410) and defaultparameter settings were used to search the Arabidopsis EST database(AAtDB 4-7) for genes that encode AP2 domain-containing proteins. Aminoacid sequence alignments were generated using the CLUSTAL W multiplesequence alignment program (Thompson, J. D., et al., 1994, Nucleic AcidsRes. 22, 4673-4680). Secondary structure predictions were based on theprinciples and software programs described by Rost (Rost, B., 1996,Methods Enzymol. 266, 525-539) and Rost and Sander (Rost, B., et al.,1993, J. Mol. Biol. 232, 589-599; Rost, B., et al., 1994, Proteins 19,55-77).

RAP2 Gene-Specific Probes. RAP2 gene-specific fragments were generatedby PCR using gene-specific primers and individual RAP2 plasmid DNAs as atemplate as specified by Perkin-Elmer (Roche Molecular Systems,Branchburg, NJ). The following primers were used to generate fragmentsrepresenting each RAP2 gene:

RAP2.1 (SEQ ID NOS:75 and 76, respectively), 5'-AAGAGGACCATCTCTCAG-3',

5'-AACACTCGCTAGCTTCTC-3';

RAP2.2 (SEQ ID NOS:77 and 78, respectively), 5'-TGGTTCAGCAGCCAACAC-3',5'-CAATGCATAGAGCTTGAGG-3';

RAP2.3 (SEQ ID NOS:100 and 101, respectively), 5'-TCATCGCCACGATCAACC-3',5'-AGCAGTCCAATGCGACGG-3';

RAP2.4 (SEQ ID NOS:79 and 80, respectively), 5'-ACGGATTTCACATCGGAG-3',5'-CTAAGCTAGAATCGAATCC-3';

RAP2.7 (SEQ ID NOS:85 and 86, respectively), 5'-CGATGGAGACGAAGACTC-3,5'-GTCGGAACCGGAGTTACC-3';

RAP2.8 (SEQ ID NOS:87 and 88, respectively), 5'-TCACTCAAAGGCCGAGATC-3',5'-TAACAACATCACCGGCTCG-3';

RAP2.9 (SEQ ID NOS:89 and 90, respectively), 5'-GTGAAGGCTTAGGAGGAG-3',5'-TGCCTCATATGAGTCAGAG-3'.

PCR-synthesized DNA fragments were gel purified and radioactivelylabeled using random oligonucleotides (Amersham) for use as probes ingene mapping and RNA gel blot experiments.

Gene Mapping Experiments. RAP2 genes were placed on the Arabidopsisgenetic map by either restriction fragment length polymorphismsegregation analysis using recombinant inbred lines as described byReiter et al. (Reiter, R. S., et al., 1992, Proc. Natl. Acad. Sci. USA89, 1477-1481) or by matrix-based analysis of pooled DNAs from theArabidopsis yUP or CIC yeast artificial chromosome (YAC) genomiclibraries (Ecker, J. R., 1990, Methods 1, 186-194; Creusot, F., et al.,1995, Plant J. 8, 763-770) using the PCR (Green, E. D., et al., 1990,Proc. Natl. Acad Sci. USA 87, 1213-1217; Kwiatkowski, T. J., et al.,1990, Nucleic Acids Res. 18, 7191-7192). Matrix based mapping resultswere confirmed by PCR using DNA from individual YAC clones.

mRNA Isolation. Polysomal poly(A) mRNAs from Arabidopsis flower, rosetteleaf, inflorescence stem internode, and hydroponically-grown roots wereisolated according to Cox and Goldberg (Cox, K. H., et al., 1988, inPlant Molecular Biology. A Practical Approach, ed. Shaw, C. H. (IRL,Oxford), pp. 1-35).

RNA Gel Blot Studies. RNA gel blot hybridizations were carried out asspecified by the manufacturer (Amersham). mRNA sizes were estimatedrelative to known RNA standards (BRL). AP2 transcripts were detectedusing a labeled DNA fragment representing nucleotides 1-1371 of the AP2cDNA plasmid clone pAP2cl (Jofuku, K. D., et al., 1994, Plant Cell 6,1211-1225).

RESULTS

The AP2 Domain Defines a Large Family of Plant Proteins. Using the AP2domain as a sequence probe 34 cDNA clones were identified that encodeputative RAP2 proteins in the Arabidopsis EST database (Materials andMethods). Several of these partial sequences have been reportedpreviously (Ohme-Takagi, et al., 1995, Plant Cell 7, 173-182; Elliot, R.C., et al., 1996, Plant Cell 8, 155-168; Klucher, K M., et al., 1996,Plant Cell 8, 137-153; Wilson, K., et al., 1996, Plant Cell 8, 659-671;Ecker, J. R., 1995, Science 268, 667-675; Weigel, D., 1995, Plant Cell7, 388-389). Based on nucleotide sequence comparison, it was inferredthat approximately half of the 34 RAP2 cDNA sequences were likely torepresent redundant clones. Therefore, a complete DNA sequence for 17putative RAP2 cDNA clones that appeared to represent unique genes andwhich contained the largest cDNA inserts was selected and generated. Itwas determined from the predicted amino acid sequences of these clonesthat the Arabidopsis RAP2 ESTs represent a minimum of 12 genes that aredesignated RAP2.1-RAP2.12. As shown in Table V, preliminary gene mappingexperiments using restriction fragment length polymorphism analysis andPCR-based screening of the Arabidopsis yUP and CIC yeast artificialchromosome libraries (Materials and Methods) revealed that at least 7members of the RAP2 gene family are distributed over 4 differentchromosomes. In addition, several family members are tightly linked inthe genome. For example, RAP2.10 is only 10 kb away from AP2, which isalso closely linked to ANT on chromosome 4 (Elliot, R. C., et al., 1996,Plant Cell 8, 155-168; Klucher, K M., et al., 1996, Plant Cell 8,137-153).

Sequence analysis also revealed that the proteins encoded by the RAP2genes are all characterized by the presence of least one AP2 domain.FIG. 4 shows a sequence comparison of 21 AP2 domains from 19 differentpolypeptides including RAP2.1-RAP2.12, AP2, ANT, TINY, and the tobaccoEREBPs. From this comparison, it was determined that there are 2conserved sequence blocks within each AP2 domain. The first block,referred to as the YRG element, consists of 19-22 amino acids, is highlybasic and contains the conserved YRG amino acid motif (FIG. 4 A and B).The second block, referred to as the RAYD (SEQ ID NO:8) element, is42-43 amino acids in length and contains a highly conserved 18-aminoacid core region that is predicted to form an amphipathic α-helix in theAP2 domains of AP2, ANT, TINY, and the EREBPs. In addition, there areseveral invariant amino acid residues within the YRG and RAYD (SEQ IDNO:8) elements that may also play a critical role in the structure orfunction of these proteins. For example, the glycine residue at position40 within the RAYD (SEQ ID NO:8) element is invariant in all AP2 domaincontaining proteins (FIG. 4 A and B) and has been shown to be importantfor AP2 function (Jofuku, K. D., et al., 1994, Plant Cell 6, 1211-1225).

                  TABLE V                                                         ______________________________________                                        Arabidopsis RAP2 genes                                                                          RAP2 gene                                                      containing YAC Chromosome                                                    Gene clones* map position†                                           ______________________________________                                        AINTEGUMENTA  ND             4-73                                               TINY ND 5-32 to 5-45                                                          RAP2.1 yUP18H2, CIC11D10 ND‡                                       RAP2.2 yUP6C1 38.sup.§                                                    yUP12G6, yUP24B8,                                                             yUP23E11, CIC4H5,                                                            RAP2.3 CIC12C2 3-21                                                           RAP2.4 CIC7D2, CIC10C4 ND‡                                         RAP2.7 yUP10E1 ND‡                                                 RAP2.8 CIC10G7 1-94 to 1-103.sup.§                                       RAP2.9 CIC9E12 1-117.sup.§                                               RAP2.10¶ ND 4-73                                                  ______________________________________                                         *YAC clones were determined to contain the specified RAP2 gene by PCRbase     DNA synthesis using genespecific primers (Green, E. D., et al., 1990,         Proc. Natl. Acad Sci. USA 87, 1213-1217; Kwiatkowski, T. J., et al., 1990     Nucleic Acid Res. 18, 7191-7192).                                             †Chromosome map positions are given with reference to the              Arabidopsis unified genetic map (AAtDB 4-7).                                  ‡YACbased map position is ambiguous.                               §Preliminary map position is based on a single contact with the          physical map.                                                            

GenBank accession numbers for complete EST sequences for RAP2 and othergenes are as follows: AINTEGUMENTA (U40256/U41339); TINY, (X94598),RAP2.1 (AF003094), RAP2.2 (AF003095), RAP2.3 (AF003096), RAP2.4(AF003097), RAP2.5 (AF003098),RAP2.6 (AF003099), RAP2.7 (AF003100),RAP2.8 (AF003101), RAP2.9 (AF003102), RAP2.10 (AF003103), RAP2.11(AF003104), and RAP2.12 (AF003105). All RAP2 cDNA clones were originallyreported with partial sequences and given GenBank accession numbers asshown in parentheses following each gene name: RAP2.1 (Z27045), RAP2.2(Z26440). RAP2.3 (TO4320 and T13104), RAP2.4 (T13774), RAP2.5 (T45365),RAP2.6 (T45770), RAP2. 7 (T2O443), RAP2.8 (Z33865), RAP2.9 (Z37270),RAP2.10 (T76017), RAP2.11 (T42962), and RAP2.12 (T42544). Due to thepreliminary nature of the EST sequence data, the predicted amino acidsequences for EST Z27045, T04320, T13774, and T42544 contained severalerrors and were incorrectly reported (Ohme-Takagi, et al., 1995, PlantCell 7, 173-182; Klucher, K M., et al., 1996, Plant Cell 8, 137-153;Wilson, K., et al., 1996, Plant Cell 8, 659-671; Ecker, J. R., 1995,Science 268, 667-675; Weigel, D., 1995, Plant Cell 7, 388-389). They arecorrectly given in the GenBank accession numbers noted above.

RAP2 cDNA sequence comparison also shows that there are at least twobranches to the RAP2 gene family tree. The AP2-like and EREBP-likebranches are distinguished by the number of AP2 domains contained withineach polypeptide and by sequences within the conserved YRG element. TheAP2-like branch of the RAP2 gene family is comprised of three genes AP2,ANT, and RAP2.7, each of which encodes a protein containing two AP2domains (FIG. 4A). In addition, these proteins possess a conservedWEAR/WESH (SEQ ID NO:14) amino acid sequence motif located in the YRGelement of both AP2 domain repeats (FIG. 4A). By contrast, genesbelonging to the EREBP-like branch of the RAP2 gene family encodeproteins with only one AP2 domain and include RAP2.1-RAP2.6,RAP2.8-RAP2.12, and TINY (FIG. 4B). Proteins in this class possess aconserved 7-amino acid sequence motif referred to as the WAAEIRD (SEQ IDNO:102) box (FIG. 4B) in place of the WEAR/WESH (SEQ ID NO:14) motiflocated in the YRG element (FIG. 4A). Based on these comparisons,separate AP2 domain consensus sequences for both classes of RAP2proteins were generated (FIG. 4 A and B). These results suggest that theAP2 domain and specific sequence elements within the AP2 domain areimportant for RAP2 protein functions.

The AP2-like class of RAP2 proteins is also characterized by thepresence of a highly conserved 25-26 amino acid linker region that liesbetween the two AP2 domain repeats (Klucher, K M., et al., 1996, PlantCell 8,137-153). This region is 40% identical and 48% similar betweenAP2, ANT and RAP2.7 and is not found in proteins belonging to theEREBP-like branch of RAP2 proteins. Molecular analysis of the ant-3mutant allele showed that the invariant C-terminal glycine residuewithin this linker region is essential for ANT function in vivo(Klucher, K M., et al., 1996, Plant Cell 8, 137-153), suggesting thatthe linker region may also play an important role in AP2 and RAP2.7function.

Sequences Within the RAYD (SEQ ID NO:8) Element are Predicted to FormAmphipathic α-Helices. As noted above, the 18-amino acid core regionwithin the RAYD (SEQ ID NO:8) element of the AP2 domain in AP2 ispredicted to form an amphipathic α-helix that may be important for AP2structure or function. Secondary structure prediction analysis was usedto determine whether this structure has been conserved in RAP2 proteins.As shown in FIG. 4, the core region represents the most highly conservedsequence block in the RAYD(SEQ ID NO:8) element of AP2 and the RAP2proteins. Secondary structure analysis predicts that all RAP2 proteinscontain sequences within the RAYD (SEQ ID NO:8) element that arepredicted to form amphipathic α-helices (FIG. 4 A and B). FIG. 4C showsthat sequences in RAP2.7-R1 are predicted to form an amphipathic α-helixthat is 100% identical to that predicted for AP2-R1 and 63% similar tothat predicted for ANT-R1. Sequences within the AP2 domain of EREBP-likeRAP2 proteins are predicted to form similar α-helical structures. FIG.4D shows that the RAP2.2, RAP2.5, and RAP2.12 α-helices are 81, 100, and81 % similar to that predicted for EREBP-3, respectively. Together,these results strongly suggest that the predicted amphipathic α-helix inthe RAYD(SEQ ID NO:8) element is a conserved structural motif that isimportant for AP2 domain function in all RAP2 proteins.

RAP2 Genes are Expressed in Floral and Vegetative Tissues. Previousstudies have shown that AP2 and ANT are differentially expressed at theRNA level during plant development (Jofuku, K. D., et al., 1994, PlantCell 6, 1211-1225; Elliot, R. C., et al., 1996, Plant Cell 8, 155-168;Klucher, K M., et al., 1996, Plant Cell 8, 137-153). AP2 is expressed atdifferent levels in developing flowers, leaves, inflorescence stems, androots. To determine where in plant development the EREBP-like class ofRAP2 genes are expressed RAP2.1, RAP2.2, RAP2.3, and RAP2.4gene-specific probes were reacted with a mRNA gel blot containingflower, leaf, inflorescence stem, and root polysomal poly(A) mRNA.Results from these experiments showed that each RAP2 gene produces auniquely sized mRNA transcript and displays a distinct pattern of geneexpression in flowers, leaves, inflorescence stems, and roots. Forexample, the RAP2.1 gene is expressed at low levels in wild-type flower,leaf, stem, and root. RAP2.2 gene expression appears to be constitutivein that RAP2.2 transcripts are detected at similar levels in wild-typeflower, leaf, stem, and root. By contrast, the RAP2.3 gene is expressedat a low level in wild-type flowers, at a slightly higher level inleaves, and is relatively highly expressed in both stems and roots.Finally, the RAP2.4 gene is also expressed in wild-type flower, leaf,stem, and root and is most highly expressed in roots and leaves. Thesedata indicate that individual members of the EREBP-like family of RAP2genes are expressed at the mRNA level in both floral and vegetativetissues and show quantitatively different patterns of gene regulation.

RAP2 Gene Expression Patterns are Affected by ap2. RAP2 gene expressionwas analyzed in ap2-10 mutant plants by RNA gel blot analysis todetermine whether AP2 is required for RAP2 gene expression. Theexpression of three RAP2 genes are differentially affected by the lossof AP2 function. For example, RAP2.2 gene expression is not dramaticallyaltered in mutant flowers, leaves, and roots compared to wild-typeLandsberg erecta but is down-regulated in mutant stem. RAP2.3 geneexpression appears unchanged in mutant roots but is up-regulated inmutant flowers and leaves and down-regulated in mutant stems. Bycontrast, RAP2.4 gene expression appears relatively unchanged in mutantstems and roots but is slightly up-regulated in mutant flowers andleaves. To control for possible secondary effects of ecotype on RAP2gene expression, RAP2 gene expression levels in wild-type C24 and ap2-10mutant stems were compared. These results show that the differences inRAP2.2 RAP2.3, and RAP2.4 gene expression in C24 and ap2-10 stem aresimilar to those observed between wild-type Landsberg erecta and ap2-10mutant stem. Together these results suggest that AP2 directly orindirectly regulates the expression of at least three RAP2 genes. Moreimportantly, these results suggest that AP2 is controlling geneexpression during both reproductive and vegetative development.

DISCUSSION

RAP2 Genes Encode a New Family of Putative DNA Binding Proteins.

One important conclusion from the characterization of these clones isthat the AP2 domain has been evolutionarily conserved in at leastArabidopsis and tobacco. In addition, there are two subfamilies of AP2domain containing proteins in Arabidopsis that are designated as theAP2-like and the EREBP-like class of RAP2 proteins. In vitro studieshave shown that both the EREBP and the AP2 proteins bind to DNA in asequence specific manner and that the AP2 domain is sufficient to conferEREBP DNA binding activity (Ohme-Takagi, et al., 1995, Plant Cell 7,173-182). From these results and the high degree of sequence similaritybetween the AP2 domain motifs in AP2, the EREBPs, and the RAP2 proteins,it is concluded that RAP2 proteins function as plant sequence specificDNA binding proteins. Although the exact amino acid residues within theAP2 domain required for DNA binding have not yet been identified,sequence comparisons have revealed two highly conserved motifs referredto as the YRG and RAYD (SEQ ID NO:8) elements within the AP2 domain.

The RAYD (SEQ ID NO:8) element is found in all known AP2 domains andcontains a conserved core region that is predicted to form anamphipathic α-helix (FIG. 4). One hypothesis for the function of thisα-helical structure is that it is involved in DNA binding, perhapsthrough the interaction of its hydrophobic face with the major groove ofDNA (Zubay, G., et al., 1959, J. Mol. Biol. 7, 1-20). Alternatively,this structure may mediate protein-protein interactions important forRAP2 functions. These interactions may involve the ability to form homo-or heterodimers similar to that observed for the MADS (SEQ ID NO:103)box family of plant regulatory proteins (Huang, H., et al., 1996, PlantCell 8, 81-94; Riechmann, J. L, et al., 1996, Proc. Natl. Acad. Sci. USA93, 4793-4798) and for the mammalian ATF/CREB family of transcriptionfactors (Hai, T., et al., 1991, Proc. Natl. Acad. Sci. USA 88,3720-3724; O'Shea, E. K, et al., 1992, Cell 68, 699-708.).

The conserved YRG element may also function in DNA binding due to thehighly basic nature of this region in all RAP2 proteins (FIG. 4).However, the YRG element also contains sequences that are specific foreach class of RAP2 protein and may be functionally important for DNAbinding. Specifically, the WAAIERD (SEQ ID NO:102) motif is highlyconserved in tobacco EREBPs and in EREBP-like RAP2 proteins. Bycontrast, the WEAR/WESH (SEQ ID NO:14) motif replaces the WAAIERD (SEQID NO:102) box in AP2-like RAP2 proteins (FIG. 4). In vitro studiessuggest that the EREBPs and AP2 recognize distinct DNA sequence elements(Ohme-Takagi, et al., 1995, Plant Cell 7, 173-182). It is possible thatthe WAAIERD (SEQ ID NO:102) and WEAR/WESH (SEQ ID NO:14) motifs may beresponsible for DNA binding sequence specificity. The presence of twoAP2 domains in AP2 may also contribute to differences in sequencespecificity. Although the molecular significance of having one or twoAP2 domain motifs is not yet known, genetic and Molecular studies haveshown that mutations in either AP2 domain affect AP2 function, implyingthat both are required for wild-type AP2 activity (Jofuku, K. D., etal., 1994, Plant Cell 6, 1211-1225).

In addition to Arabidopsis and tobacco, cDNAs that encode diverse AP2domain-containing proteins have been found in maize, rice, castor bean,and several members of the Brassicaceae including canola (Ohme-Takagi,et al., 1995, Plant Cell 7, 173-182; Elliot, R. C., et al., 1996, PlantCell 8, 155-168; Klucher, K M., et al., 1996, Plant Cell 8, 137-153;Wilson, K., et al., 1996, Plant Cell 8, 659-671 and Weigel, D., 1995,Plant Cell 7, 388-389). This strongly suggests that the AP2 domain is animportant and evolutionarily conserved element necessary for thestructure or function of these proteins.

RAP2 Gene Expression in Floral and Vegetative Tissues. The AP2, RAP2.1,RAP2.2, RAP2.3, and RAP2.4 genes show overlapping patterns of geneexpression at the mRNA level in flowers, leaves, inflorescence stems,and roots. However, each gene appears to be differentially regulated interms of its mRNA prevalence. The overlap in RAP2 gene activity couldaffect the genetic analysis of AP2 and RAP2 gene functions if thesegenes are also functionally redundant. For example, in flowerdevelopment AP2 and ANT show partially overlapping patterns of geneexpression at the organ and tissue levels (Jofuku, K. D., et al., 1994,Plant Cell 6, 1211-1225; Elliot, R. C., et al., 1996, Plant Cell 8,155-168; Klucher, K M., et al., 1996, Plant Cell 8, 137-153; W. Szeto).From single and double mutant analysis it has also been suggested thatAP2 may be partially redundant in function with ANT (Elliot, R. C., etal., 1996, Plant Cell 8, 155-168). The phenomenon of genetic redundancyand its ability to mask the effects of gene mutation is more clearlydemonstrated by the MADS (SEQ ID NO:103) domain containing floralregulatory genes APETALA1 (AP1) and CAULIFLOWER (CAL). Genetic studieshave demonstrated that mutations in cal show no visible floral phenotypeexcept when in double mutant combination with ap1(Bowman, J. L, et al.,1993, Development Cambridge, U.K, 119, 721-743), indicating that AP1 iscompletely redundant in function for CAL. The hypothesis that the RAP2genes may have genetically redundant functions is supported by the factthat the dominant gain-of-function mutation tiny is the only ArabidopsisRAP2 EREBP-like gene mutant isolated to date (Wilson, K., et al., 1996,Plant Cell 8, 659-671).

AP2 Activity Is Detectable in Vegetative Development. The presentanalysis of RAP2 gene expression in wild-type and ap2-10 plants suggeststhat AP2 contributes to the regulation of RAP2 gene activity throughoutArabidopsis development. RAP2 gene expression is both positively andnegatively affected by the absence of AP2 activity during development.The observed differences in RAP2.2, RAP2.3, and RAP2.4 gene expressionlevels in wild-type and ap2-10 flowers and vegetative tissues are notapparently due to differences in ecotype because similar changes in geneexpression levels were observed for all three RAP2 genes in stems whenecotype was controlled. The regulation of RAP2 gene expression by AP2 instems clearly indicates that unlike other floral homeotic genes AP2functions in both reproductive and vegetative development.

EXAMPLE 5

This example shows that transgenic plants of the invention bear seedwith altered fatty acid content and composition.

Antisense transgenic plants were prepared using AP2, RAP2.8, and RAP2.1(two independent plants) using methods described above. The fatty acidcontent and composition were determined using gas chromatography asdescribed Broun and Somerville Plant Physiol. 113:933-942(1997). Theresults are shown in Table VI (for AP2) and Table VII (for the RAP2genes). As can be seen there, the transgenic plants of the inventionhave increased fatty acid content as compared to wild-type plants. Inaddition, the profile of fatty acids is altered in the plants.

                                      TABLE VI                                    __________________________________________________________________________    Analysis of Arabidopsis seed fatty acid content and composition.                            Total Fatty                                                        Seed Mass Acid Content Fatty acid Methyesters (%) (area % by GLC)          Plants   (μg/seed)                                                                       (μg/seed)                                                                        16:0                                                                             18:0                                                                             18:1                                                                             18:2                                                                             18:3                                                                             20:1                                                                             22:1                                                                             24:0                                 __________________________________________________________________________    Mutant   27   3.94  9.4                                                                              4.4                                                                              2.5                                                                              70.8                                                                             0.8                                                                              6.6                                                                              0.1                                                                              1.9                                    ap2-10                                                                        aAP2 transgenic                                                               plants                                                                        C24 15-522 (F1-1) 34 11.0 4.5 3.3 3.1 49.0 3.2 26.3 0.6 4.9                   C34 15-542 (F1-2) 24 2.5 6.3 3.4 3.1 52.1 4.0 28.1 0.4 0.4                    Wild-type 20 2.67 9.4 4.9 8.8 62.7 0.7 8.1 0.3 1.9                            C24                                                                         __________________________________________________________________________

                                      TABLE VII                                   __________________________________________________________________________    Analysis of Arabidopsis RAP2 antisense transgenic seed fatty acid content     and composition.                                                                            Total Fatty                                                                           Seed Mass Acid Content Fatty acid methyesters (%)                           (area % by GLC)                                           Plants   (μg/seed)                                                                       (μg/seed)                                                                        16:0                                                                             18:0                                                                             18:1                                                                             18:2                                                                             18:3                                                                             20:1                                                                             22:1                                                                             24:0                                 __________________________________________________________________________    RAP2 antisense                                                                  transgenic plants                                                             RAP34-2.8C LE 24 9.7 5.1 3.0 3.6 53.6 3.2 29.3 0.3 0.2                        RAP34-2.1A' COL 34 11.0 9.5 1.9 3.0 53.6 4.2 20.3 5.5 0.5                     RAP34-2.1D' COL 26 8.1 4.6 2.6 2.6 50.3 3.2 33.2 0.3 1.0                      Wild-type 24 ND 6.0 4.0 14.0 27.0 18.0 22.0 2.0 ND                            COL.sup.a                                                                   __________________________________________________________________________     ND, not determined                                                            .sup.a Papatak et al. (1994) Oil content and fatty acid composition of        seeds of various ecotypes of Arabidopsis thaliana: a search for useful        genetic variants. Curr. Sci. 67, 470-472.                                

EXAMPLE 6

This example describes construction of promoter construct which are usedto prepare expression cassettes useful in making transgenic plants ofthe invention. In particular, this example shows use of two preferredpromoters, the promoter from the AP2 gene and the promoter from the Bel1gene.

FIG. 5 shows a AP2 promoter construct. pAP2 represents the 16.3 kb AP2promoter vector cassette that is used to generate chimeric genes for usein plant transformations described here. pAP2 is comprised of the 4.0 kbpromoter region of the Arabidopsis AP2 gene. The Ti plasmid vector usedis pDE1000 vector (Plant Genetic Systems, Ghent, Belgium). The pDE1000vector DNA was linearized with BamH1 and the AP2 promoter regioninserted as a 4.0 kb BamH1 DNA fragment from plasmid subclone pLE7.2. Atthe 3' end of the inserted AP2 promoter region, designated AP2, liethree restriction sites (EcoR1, Sma1 and SnaB1) into which differentgene coding regions can be inserted to generate chimeric AP2promoter/gene cassettes. NOS::NPTII represents the plant selectablemarker gene NPTII under the direction of the nopaline synthase promoterwhich confers resistance to the antibiotic kanamycin to transformedplants cells carrying an integrated AP2 promoter cassette. LB and RBrepresent the T-DNA left and right border sequences, respectively, thatare required for transfer of T-DNA containing the AP2 promoter cassetteinto the plant genome. PVS1 designates the bacterial DNA sequences thatfunction as a bacterial origin of replication in both E. coli andAgrobacterium tumefaciens, thus allowing pAP2 plasmid replication andretention in both bacteria. Amp^(R) and Sm/Sp^(R) designate bacterialselectable marker genes that confer resistance to the antibioticsampicillin and streptomycin/spectinomycin, respectively, and allows forselection of Agrobacterium strains that carry the pAP2 recombinantplasmid.

FIG. 6 shows a BEL1 promoter construct. pBEL1 represents the 16.8 kbBEL1 promoter vector cassette that is used to generate chimeric genesfor use in plant transformations described here. pBEL1 is comprised ofthe 4.5 kb promoter region of the Arabidopsis BEL1 gene. The Ti plasmidvector used is pDE1000 vector (Plant Genetic Systems, Ghent, Belgium).The pDE1000 vector DNA was linearized will BamH1 and the BEL1 promoterregion inserted as a 4.5 kb BamH1-Bg12 DNA fragment from plasmidsubclone pλ1C9R (L. Reiser, unpublished). At the 3' end of the insertedBEL1 promoter region, designated BEL1, lie three restriction sites(EcoR1, Sma1 and SnaB1) into which different gene coding regions can beinserted to generate chimeric BEL promoter/gene cassettes. NOS::NPTIIrepresents the plant selectable marker gene NPTII under the direction ofthe nopaline synthase promoter which confers resistance to theantibiotic kanamycin to transformed plants cells carrying an integratedBEL promoter cassette. LB and RB represent the T-DNA left and rightborder sequences, respectively, that are required for transfer of T-DNAcontaining the BEL promoter cassette into the plant genome. PVS1designates the bacterial DNA sequences that function as a bacterialorigin of replication in both E. coli and Agrobacterium tumefaciens,thus allowing pBEL plasmid replication and retention in both bacteria.Amp^(R) and Sm/Sp^(R) designate bacterial selectable marker genes thatconfer resistance to the antibiotics ampicillin andstreptomycin/spectinomycin, respectively, and allows for selection ofAgrobacterium strains that carry the pBEL recombinant plasmid.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 103                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 559 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..559                                                          (D) OTHER INFORMATION: - #/note= "canola APETALA2 (AP2)           domain                                                                                         containing - #(ADC) gene"                                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: - #/note= "exon 2"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 27..169                                                         (D) OTHER INFORMATION: - #/note= "intron 2"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 170..200                                                        (D) OTHER INFORMATION: - #/note= "exon 3"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 201..306                                                        (D) OTHER INFORMATION: - #/note= "intron 3"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 307..394                                                        (D) OTHER INFORMATION: - #/note= "exon 4"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 395..473                                                        (D) OTHER INFORMATION: - #/note= "intron 4"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 474..559                                                        (D) OTHER INFORMATION: - #/note= "exon 5"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GGACTGTGGG AAACAAGTTT ACTTAGGTAT GATCATGTAA TGTTGTTCAA AC -            #ACAGATCA     60                                                                 - - AATATCCTAT TGAAACTAAG TTGTGTTGTG TCCGTCCATT TTTATATGAT TT -            #CTTCGACC    120                                                                 - - AAATAAAGGT TTNANTATCT CCTTATATTA CTTTTTGTTA CATATTCAGG TG -            #GATTTGAC    180                                                                 - - ACAGCACATG CCGCTGCTCG GTATGTTTAA CTCATCCAAA TATGATCAAT TA -            #GAACGAAT    240                                                                 - - CTAATATTCC TTATTTGTAA TTTGCTGATA TACAAATTAA TTTGGGTGGG TA -            #ACTGTTTG    300                                                                 - - GGACAGTGCC TACGATAGAG CCGCAGTTAA GTTTAGAGGT GTNATGCAGA AT -            #ATNANTTN    360                                                                 - - CAANATTGAA GACTANGTGG AGGANTTGAA ACAGGTAGAA CATCTATCAT TT -            #GGTNGGNC    420                                                                 - - CAAGCNNTNG ACCNANATCT TACTCCACCN TCCNCCTNTA ATNCNTGNTG CN -            #GATGAGCA    480                                                                 - - CCTTGACAAN GGACGAGTCA TGCNTGTCAT TAGNCGCNAN CCCTGGGTTC CA -            #NNAAGCNC    540                                                                 - - CTCAAGTANA GANGGTCAC             - #                  - #                      - #559                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 291 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..291                                                          (D) OTHER INFORMATION: - #/note= "soybean APETALA2 (AP2)           domain                                                                                         containing - #(ADC) gene partial sequence                                     generated - #by sequencing from the 5' end"                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 1..27                                                           (D) OTHER INFORMATION: - #/note= "exon 2"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 28..264                                                         (D) OTHER INFORMATION: - #/note= "intron 2"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 265..291                                                        (D) OTHER INFORMATION: - #/note= "exon 3"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GGATTGTGGG AAACAAGTTT ATCTAGGTAA AGTTGAYTAA YAACAATAAT TG -            #TATATGTG     60                                                                 - - TTTGTGAGAA CTGTGGCAST TATTTTCCCT AATTATCGTT TTAAGACGCT AA -            #MACGGTTT    120                                                                 - - TTTTYCCCTT GTCTTGTGTT TTTTGYCTTG GCTGTGAYGC GGTAAAMACA AN -            #AGTGTGAG    180                                                                 - - TGTGTGTTGT GTGTGGGTGA GGAYTYTTTC CTNTCNCCGT GGTGACTGAC TT -            #GATGGNTC    240                                                                 - - TTGNCTGGGT NCANNTTNTC TACGTGGATT CGACACNNCA CATGCGGCNG C - #                291                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 972 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..972                                                          (D) OTHER INFORMATION: - #/note= "soybean APETALA2 (AP2)           domain                                                                                         containing - #(ADC) gene partial sequence                                     generated - #by sequencing from the 3' end"                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 629..716                                                        (D) OTHER INFORMATION: - #/note= "exon 4"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 717..876                                                        (D) OTHER INFORMATION: - #/note= "intron 5"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                            (B) LOCATION: 877..972                                                        (D) OTHER INFORMATION: - #/note= "exon 5"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - NCGGGACGGA CNGGNCGGAN GCNANGCGCN NNNAGACGGN ANCGCAANNA CG -            #NCNCNTCG     60                                                                 - - CCTCCGNGGG CCCNACNCNC TCGGACGGNT GNGAGGNCCC CNNGNNTCNN GC -            #NNGGNAGC    120                                                                 - - ANGNGGTGCN GCCNTGGCGA NCCGCCCGGN NGAGNGNAGN CNTGGNGNCG AC -            #ACCNTGCA    180                                                                 - - GNCNTCTNGC NATGNGATGG NNAACNGGAG ACCGGAGTGA CNGTCGNGAG NA -            #ANGCGANA    240                                                                 - - NNTNNTGGTG NTCCCGGGCG NGAANGNCCN GACAGATGGG TGGAACGTAT TG -            #CAGTGTAN    300                                                                 - - ACCAAGAAGG GTNGGACGGC GTATTTCTAA TGTTAGGNTA NNNTTTNTCC TT -            #TGGTTANT    360                                                                 - - CTGCCNGNGC GAAACNGGGG AGATGGNNNG GGGNGAGATT TTTNTNTGNG NG -            #ACGACTGA    420                                                                 - - NGNNTCTGTT GGGTNAATTG TCTAGGGGAT TGACACNCAC ATGCGTGCTC GT -            #GGCCCTGC    480                                                                 - - CCCTTCCTTC AGTATNATAC CCAAGCTTGT ATNTTACTTT NTCCATGTCT TG -            #AACCAAAT    540                                                                 - - ATCAAATATT ATTGTNAATC ACATTTCGTT GTGGNCCGGG AATTGTGAGT CT -            #CAAAGAAA    600                                                                 - - ATTGTGTATT NTCCGTCTCT CTTTTCAGTG CTTATGATAG AGCGGCTATT AA -            #ATTCCGAG    660                                                                 - - GAGTGGAGGC TGACATTAAC TTCAATATTG NAGACTATGA AGATGACTTG AA -            #GCAGGTGA    720                                                                 - - TCAATTTGTG GATTATGTTT TTTTTATTCG AATAAATGCA TTTATCGTAT TT -            #ATCTTATC    780                                                                 - - TTACAGTCAT ACGTATAGGA TGCACCTTAT CTCCCACAGT TAGTGTTTTT TT -            #TATCTGAA    840                                                                 - - TTATTCTCAT GATTTTGTTA AATGCAATGT TAATAGATGA GCAATCTTAC CA -            #AGGAAGAG    900                                                                 - - TTCGTCCACG TGCTTCGCCG CCAAAGCACT GGATTTCCGA GAGGAAGCTC CA -            #AGTATAGA    960                                                                 - - GGTCACTTTG CA              - #                  - #                      - #      972                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..67                                                           (D) OTHER INFORMATION: - #/note= "AP2-R1 direct repeat at                          positions - #129 to 195"                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 32..49                                                          (D) OTHER INFORMATION: - #/note= "putative AP2-R1 amphipathic                      alpha-helix - #(SEQ ID NO:6)"                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ser Ser Gln Tyr Arg Gly Val Thr Phe Tyr Ar - #g Arg Thr Gly Arg Trp      1               5   - #                10  - #                15               - - Glu Ser His Ile Trp Asp Cys Gly Lys Gln Va - #l Tyr Leu Gly Gly Phe                  20      - #            25      - #            30                   - - Thr Asp Ala His Ala Ala Ala Arg Ala Tyr As - #p Arg Ala Ala Ile Lys              35          - #        40          - #        45                       - - Phe Arg Gly Val Glu Ala Asp Ile Asn Phe As - #n Ile Asp Asp Tyr Asp          50              - #    55              - #    60                           - - Asp Asp Leu                                                              65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "AP2-R2 direct repeat at                          positions - #221 to 288"                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 33..50                                                          (D) OTHER INFORMATION: - #/note= "putative AP2-R2 amphipathic                      alpha-helix - #(SEQ ID NO:7)"                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Ser Ser Lys Tyr Arg Gly Val Thr Leu His Ly - #s Cys Gly Arg Trp Glu      1               5   - #                10  - #                15               - - Ala Arg Met Gly Gln Phe Leu Gly Lys Lys Ty - #r Val Tyr Leu Gly Leu                  20      - #            25      - #            30                   - - Phe Asp Thr Glu Val Glu Ala Ala Arg Ala Ty - #r Asp Lys Ala Ala Ile              35          - #        40          - #        45                       - - Lys Cys Asn Gly Lys Asp Ala Val Thr Asn Ph - #e Asp Pro Ser Ile Tyr          50              - #    55              - #    60                           - - Asp Glu Glu Leu                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "putative AP2-R1 amphipathic                      alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Phe Asp Thr Ala His Ala Ala Ala Arg Ala Ty - #r Asp Arg Ala Ala Ile      1               5   - #                10  - #                15               - - Lys Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "putative AP2-R2 amphipathic                      alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Phe Asp Thr Glu Val Glu Ala Ala Arg Ala Ty - #r Asp Lys Ala Ala Ile      1               5   - #                10  - #                15               - - Lys Cys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Arg Ala Tyr Asp                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..77                                                           (D) OTHER INFORMATION: - #/note= "ANT-R1 direct repeat"              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 44..59                                                          (D) OTHER INFORMATION: - #/note= "putative ANT-R1 amphipathic                      alpha-helix - #(SEQ ID NO:37)"                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Thr Ser Gln Tyr Arg Gly Val Thr Arg His Ar - #g Trp Thr Gly Arg Tyr      1               5   - #                10  - #                15               - - Glu Ala His Leu Trp Asp Asn Ser Phe Lys Ly - #s Glu Gly His Ser Arg                  20      - #            25      - #            30                   - - Lys Gly Arg Gln Val Tyr Leu Gly Gly Tyr As - #p Met Glu Glu Lys Ala              35          - #        40          - #        45                       - - Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Ty - #r Trp Gly Pro Ser Thr          50              - #    55              - #    60                           - - His Thr Asn Phe Ser Ala Glu Asn Tyr Gln Ly - #s Glu Ile                  65                  - #70                  - #75                               - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..69                                                           (D) OTHER INFORMATION: - #/note= "ANT-R2 direct repeat"              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 37..51                                                          (D) OTHER INFORMATION: - #/note= "putative ANT-R2 amphipathic                      alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Ala Ser Ile Tyr Arg Gly Val Thr Arg His Hi - #s Gln His Gly Arg Trp      1               5   - #                10  - #                15               - - Gln Ala Arg Ile Gly Arg Val Ala Gly Asn Ly - #s Asp Leu Tyr Leu Gly                  20      - #            25      - #            30                   - - Thr Phe Gly Thr Gln Glu Glu Ala Ala Glu Al - #a Tyr Asp Val Ala Ala              35          - #        40          - #        45                       - - Ile Lys Phe Arg Gly Thr Asn Ala Val Thr As - #n Phe Asp Ile Thr Arg          50              - #    55              - #    60                           - - Tyr Asp Val Asp Arg                                                      65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..67                                                           (D) OTHER INFORMATION: - #/note= "RAP2.7-R1 direct repeat"           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 33..49                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.7-R1 amphipathi                    alpha hel - #ix (SEQ ID NO:36)"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Ser Ser Gln Tyr Arg Gly Val Thr Phe Tyr Ar - #g Arg Thr Gly Arg Trp      1               5   - #                10  - #                15               - - Glu Ser His Ile Trp Asp Cys Gly Lys Gln Va - #l Tyr Leu Gly Gly Phe                  20      - #            25      - #            30                   - - Asp Thr Ala His Ala Ala Ala Arg Ala Tyr As - #p Arg Ala Ala Ile Lys              35          - #        40          - #        45                       - - Phe Arg Gly Val Asp Ala Asp Ile Asn Phe Th - #r Leu Gly Asp Tyr Glu          50              - #    55              - #    60                           - - Glu Asp Met                                                              65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..53                                                           (D) OTHER INFORMATION: - #/note= "RAP2.7-R2 direct repeat"           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 26..34                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.7-R2 amphipathi    c                                                                                             alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Ser Ser Lys Tyr Arg Gly Val Thr Leu His Ly - #s Cys Gly Arg Trp Glu      1               5   - #                10  - #                15               - - Ala Arg Met Gly Gln Phe Leu Gly Lys Lys Al - #a Tyr Asp Lys Ala Ala                  20      - #            25      - #            30                   - - Ile Asn Thr Asn Gly Arg Glu Ala Val Thr As - #n Phe Glu Met Ser Ser              35          - #        40          - #        45                       - - Tyr Gln Asn Glu Ile                                                          50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Tyr Arg Gly Val Thr                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Ala or Ser"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Arg or His"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Gly Arg Trp Glu Xaa Xaa                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Val Tyr Leu Gly                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Ala Ala Ile Lys                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..69                                                           (D) OTHER INFORMATION: - #/note= "AP2 domain within tobacco                        EREBP-1"                                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 36..51                                                          (D) OTHER INFORMATION: - #/note= "putative EREBP-1 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Gly Arg His Tyr Arg Gly Val Arg Arg Arg Pr - #o Trp Gly Lys Phe Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Ala Lys Asn Gly Al - #a Arg Val Trp Leu Gly                  20      - #            25      - #            30                   - - Thr Tyr Glu Thr Asp Glu Glu Ala Ala Ile Al - #a Tyr Asp Lys Ala Ala              35          - #        40          - #        45                       - - Tyr Arg Met Arg Gly Ser Lys Ala His Leu As - #n Phe Pro Leu Glu Val          50              - #    55              - #    60                           - - Ala Asn Phe Lys Gln                                                      65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..69                                                           (D) OTHER INFORMATION: - #/note= "AP2 domain within tobacco                        EREBP-2"                                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..51                                                          (D) OTHER INFORMATION: - #/note= "putative EREBP-2 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Gly Arg His Tyr Arg Gly Val Arg Gln Arg Pr - #o Trp Gly Lys Phe Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Ala Lys Asn Gly Al - #a Arg Val Trp Leu Gly                  20      - #            25      - #            30                   - - Thr Tyr Glu Thr Ala Glu Glu Ala Ala Leu Al - #a Tyr Asp Lys Ala Ala              35          - #        40          - #        45                       - - Tyr Arg Met Arg Gly Ser Lys Ala Leu Leu As - #n Phe Pro His Arg Ile          50              - #    55              - #    60                           - - Gly Leu Asn Glu Pro                                                      65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "AP2 domain within tobacco                        EREBP-3"                                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..50                                                          (D) OTHER INFORMATION: - #/note= "putative EREBP-3 amphipathic                     alpha-helix - #(SEQ ID NO:41)"                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Glu Val His Tyr Arg Gly Val Arg Lys Arg Pr - #o Trp Gly Arg Tyr Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Gly Lys Lys Ser Ar - #g Val Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Asp Thr Ala Glu Glu Ala Ala Lys Ala Ty - #r Asp Thr Ala Ala Arg              35          - #        40          - #        45                       - - Glu Phe Arg Gly Pro Lys Ala Lys Thr Asn Ph - #e Pro Ser Pro Thr Glu          50              - #    55              - #    60                           - - Asn Gln Ser Pro                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..69                                                           (D) OTHER INFORMATION: - #/note= "AP2 domain within tobacco                        EREBP-4"                                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..51                                                          (D) OTHER INFORMATION: - #/note= "putative EREBP-4 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Lys Lys His Tyr Arg Gly Val Arg Gln Arg Pr - #o Trp Gly Lys Phe Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Asn Arg Lys Gly Th - #r Arg Val Trp Leu Gly                  20      - #            25      - #            30                   - - Thr Phe Asp Thr Ala Ile Glu Ala Ala Lys Al - #a Tyr Asp Arg Ala Ala              35          - #        40          - #        45                       - - Phe Lys Leu Arg Gly Ser Lys Ala Ile Val As - #n Phe Pro His Arg Ile          50              - #    55              - #    60                           - - Gly Leu Asn Glu Pro                                                      65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.2                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 34..51                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.2 amphipathic                     alpha-helix - #(SEQ ID NO:38)"                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Lys Asn Gln Tyr Arg Gly Ile Arg Gln Arg Pr - #o Trp Gly Lys Trp Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Arg Lys Gly Ser Ar - #g Glu Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Asp Thr Ala Glu Glu Ala Ala Arg Ala Ty - #r Asp Ala Ala Ala Arg              35          - #        40          - #        45                       - - Arg Ile Arg Gly Thr Lys Ala Lys Val Asn Ph - #e Pro Glu Glu Lys Asn          50              - #    55              - #    60                           - - Pro Ser Val Val                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.3                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 36..50                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.3 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Lys Asn Val Tyr Arg Gly Ile Arg Lys Arg Pr - #o Trp Gly Lys Trp Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Arg Lys Gly Val Ar - #g Val Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Asn Thr Ala Glu Glu Ala Ala Met Ala Ty - #r Asp Val Ala Ala Lys              35          - #        40          - #        45                       - - Gln Ile Arg Gly Asp Lys Ala Lys Leu Asn Ph - #e Pro Asp Leu His His          50              - #    55              - #    60                           - - Pro Pro Pro Pro                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.5                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..50                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.5 amphipathic                     alpha-helix - #(SEQ ID NO:39)"                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Glu Ile Arg Tyr Arg Gly Val Arg Lys Arg Pr - #o Trp Gly Arg Tyr Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro Gly Lys Lys Thr Ar - #g Val Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Asp Thr Ala Glu Glu Ala Ala Arg Ala Ty - #r Asp Thr Ala Ala Arg              35          - #        40          - #        45                       - - Asp Phe Arg Gly Ala Lys Ala Lys Thr Asn Ph - #e Pro Thr Phe Leu Glu          50              - #    55              - #    60                           - - Leu Ser Asp Gln                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.6                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 34..51                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.6 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Pro Lys Lys Tyr Arg Gly Val Arg Gln Arg Pr - #o Trp Gly Lys Trp Ala      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Asp Pro His Lys Ala Thr Ar - #g Val Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Glu Thr Ala Glu Ala Ala Ala Arg Ala Ty - #r Asp Ala Ala Ala Leu              35          - #        40          - #        45                       - - Arg Phe Arg Gly Ser Lys Ala Lys Leu Asn Ph - #e Pro Glu Asn Val Gly          50              - #    55              - #    60                           - - Thr Gln Thr Ile                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.12                                                                                        AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 34..51                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.12                                 amphipathic - #alpha-helix (SEQ ID NO:40)"                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Lys Asn Gln Tyr Arg Gly Ile Arg Gln Arg Pr - #o Trp Gly Lys Trp        Ala                                                                             1               5   - #                10  - #                15              - - Ala Glu Ile Arg Asp Pro Arg Glu Gly Ala Ar - #g Ile Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Lys Thr Ala Glu Glu Ala Ala Arg Ala Ty - #r Asp Ala Ala Ala Arg              35          - #        40          - #        45                       - - Arg Ile Arg Gly Ser Lys Ala Lys Val Asn Ph - #e Pro Glu Glu Asn Met          50              - #    55              - #    60                           - - Lys Ala Asn Ser                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis TINY                      AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..50                                                          (D) OTHER INFORMATION: - #/note= "putative TINY amphipathic                        alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - His Pro Val Tyr Arg Gly Val Arg Lys Arg As - #n Trp Gly Lys Trp Val      1               5   - #                10  - #                15               - - Ser Glu Ile Arg Glu Pro Arg Lys Lys Ser Ar - #g Ile Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Pro Ser Pro Glu Met Ala Ala Arg Ala Hi - #s Asp Val Ala Ala Leu              35          - #        40          - #        45                       - - Ser Ile Lys Gly Ala Ser Ala Ile Leu Asn Ph - #e Pro Asp Leu Ala Gly          50              - #    55              - #    60                           - - Ser Phe Pro Arg                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.1                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..50                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.1 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Arg Lys Pro Tyr Arg Gly Ile Arg Arg Arg Ly - #s Trp Gly Lys Trp Val      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Glu Pro Asn Lys Arg Ser Ar - #g Leu Trp Leu Gly Ser                  20      - #            25      - #            30                   - - Tyr Thr Thr Asp Ile Ala Ala Ala Arg Ala Ty - #r Asp Val Ala Val Phe              35          - #        40          - #        45                       - - Tyr Leu Arg Gly Pro Ser Ala Arg Leu Asn Ph - #e Pro Asp Leu Leu Leu          50              - #    55              - #    60                           - - Gln Glu Glu Asp                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.4                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 35..50                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.4 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Thr Lys Leu Tyr Arg Gly Val Arg Gln Arg Hi - #s Trp Gly Lys Trp Val      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Leu Pro Arg Asn Arg Thr Ar - #g Leu Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Asp Thr Ala Glu Glu Ala Ala Leu Ala Ty - #r Asp Lys Ala Ala Tyr              35          - #        40          - #        45                       - - Lys Leu Arg Gly Asp Phe Ala Arg Leu Asn Ph - #e Pro Asn Leu Arg His          50              - #    55              - #    60                           - - Asn Gly Phe His                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..66                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.8                                                                                         AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 33..48                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.8 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Ser Ser Lys Tyr Lys Gly Val Val Pro Gln Pr - #o Asn Gly Arg Trp Gly      1               5   - #                10  - #                15               - - Ala Gln Ile Tyr Glu Lys His Gln Arg Val Tr - #p Leu Gly Thr Phe Asn                  20      - #            25      - #            30                   - - Glu Gln Glu Glu Ala Ala Arg Ser Tyr Asp Il - #e Ala Ala Cys Arg Phe              35          - #        40          - #        45                       - - Arg Gly Arg Asp Ala Val Val Asn Phe Lys As - #n Val Leu Glu Asp Gly          50              - #    55              - #    60                           - - Asp Leu                                                                  65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.10                                                                                        AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 36..51                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.10 amphipathic                    alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Asp Lys Pro Tyr Lys Gly Ile Arg Met Arg Ly - #s Trp Gly Lys Trp Val      1               5   - #                10  - #                15               - - Ala Glu Ile Arg Glu Pro Asn Lys Arg Ser Ar - #g Ile Trp Leu Gly Ser                  20      - #            25      - #            30                   - - Tyr Ser Thr Pro Glu Ala Ala Ala Arg Ala Ty - #r Asp Thr Ala Val Phe              35          - #        40          - #        45                       - - Tyr Leu Arg Gly Pro Ser Ala Arg Leu Asn Ph - #e Pro Glu Leu Leu Ala          50              - #    55              - #    60                           - - Gly Val Thr Val                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..68                                                           (D) OTHER INFORMATION: - #/note= "EREBP-like Arabidopsis           RAP2.11                                                                                        AP2 domai - #n"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 34..50                                                          (D) OTHER INFORMATION: - #/note= "putative RAP2.11 amphipathic                    alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Lys Thr Lys Phe Val Gly Val Arg Gln Arg Pr - #o Ser Gly Lys Trp Val      1               5   - #                10  - #                15               - - Ala Glu Ile Lys Asp Thr Thr Gln Lys Ile Ar - #g Met Trp Leu Gly Thr                  20      - #            25      - #            30                   - - Phe Glu Thr Ala Glu Glu Ala Ala Arg Ala Ty - #r Asp Glu Ala Ala Cys              35          - #        40          - #        45                       - - Leu Leu Arg Gly Ser Asn Thr Arg Thr Asn Ph - #e Ala Asn His Phe Pro          50              - #    55              - #    60                           - - Asn Asn Ser Gln                                                          65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Val or Ile"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Tyr Arg Gly Xaa Arg                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = positively charged                                           amino aci - #d"                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Trp, Phe or Tyr"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Ala or Val"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Arg or Lys"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Asp or Glu"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Trp Gly Xaa Xaa Xaa Ala Glu Ile Xaa Xaa                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Ser or Thr"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Tyr or Phe"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Trp Leu Gly Xaa Xaa                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = Ile, Leu or positively                                       charged a - #mino acid"                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Glu Glu Ala Ala Xaa Ala Tyr Asp                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: - #/note= "putative RAP2.7-R1 amphipathi    c                                                                                             alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Asp Thr Ala His Ala Ala Ala Arg Ala Tyr As - #p Arg Ala Ala Ile Lys      1               5   - #                10  - #                15               - - Phe                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/note= "putative ANT-R1 amphipathic                      alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Met Glu Glu Lys Ala Ala Arg Ala Tyr Asp Le - #u Ala Ala Leu Lys Tyr      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "putative RAP2.2 amphipathic                      alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Asp Thr Ala Glu Glu Ala Ala Arg Ala Tyr As - #p Ala Ala Ala Arg Arg      1               5   - #                10  - #                15               - - Ile Arg                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/note= "putative RAP2.5 amphipathic                      alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Thr Ala Glu Glu Ala Ala Arg Ala Tyr Asp Th - #r Ala Ala Arg Asp Phe      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "putative RAP2.12 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Lys Thr Ala Glu Glu Ala Ala Arg Ala Tyr As - #p Ala Ala Ala Arg Arg      1               5   - #                10  - #                15               - - Ile Arg                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/note= "putative EREBP-3 amphipathic                     alpha-helix"                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - Thr Ala Glu Glu Ala Ala Lys Ala Tyr Asp Th - #r Ala Ala Arg Glu Phe      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..25                                                           (D) OTHER INFORMATION: - #/note= "AP2 linker region"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - Lys Gln Met Thr Asn Leu Thr Lys Glu Glu Ph - #e Val His Val Leu Arg      1               5   - #                10  - #                15               - - Arg Gln Ser Thr Gly Phe Pro Arg Gly                                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: - #/note= "ANT linker region"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - Glu Asp Met Met Lys Asn Met Thr Arg Gln Gl - #u Tyr Val Ala His Leu      1               5   - #                10  - #                15               - - Arg Arg Lys Ser Ser Gly Phe Ser Arg Gly                                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: - #/note= "RAP2.7 linker region"              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - Met Lys Gln Val Gln Asn Leu Ser Lys Glu Gl - #u Phe Val His Ile Leu      1               5   - #                10  - #                15               - - Arg Arg Gln Ser Thr Gly Phe Ser Arg Gly                                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/product= "OTHER"                                        /note= - #"Xaa = positively charged                                           amino aci - #d"                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - Asn Leu Thr Xaa Glu Glu Phe Val His                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - Leu Arg Arg Gln Ser Thr Gly Phe Ser Arg Gl - #y                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "JOAP2U primer"                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - GTTGCCGCTG CCGTAGTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JOAP2L primer"                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - GGTTCATCCT GAGCCGCATA TC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.1U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - CTCAAGAAGA AGTGCCTAAC CACG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.1L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - GCAGAAGCTA GAAGAGCGTC GA           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.2U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - GGAAAATGGG CTGCGGAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.2L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - GTTACCTCCA GCATCGAACG AG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.4U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - GCTGGATCTT GTTTCGCTTA CG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.4L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - GCTTCAAGCT TAGCGTCGAC TG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..20                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.5U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - AGATGGGCTT GAAACCCGAC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.5L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - CTGGCTAGGG CTACGCGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.6U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - TTCTTTGCCT CCTCAACCAT TG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.6L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - TCTGAGTTCC AACATTTTCG GG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.7U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - GAAATTGGTA ACTCCGGTTC CG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.7L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - CCATTTTGCT TTGGCGCATT AC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.8U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - GGCGTTACGC CTCTACCGG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..20                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.8L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - CGCCGTCTTC CAGAACGTTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.9U primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - ATCACGGATC TGGCTTGGTT C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.9L primer"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - GCCTTCTTCC GTATCAACGT CG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.10U primer"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - GTCAACTCCG GCGGTTACG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.10L primer"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - TCTCCTTATA TACGCCGCCG A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.11U primer"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - GAGAAGAGCA AAGGCAACAA GAC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.11L primer"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - AGTTGTTAGG AAAATGGTTT GCG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..25                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.12U primer"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - AAACCATTCG TTTTCACTTC GACTC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..25                                                           (D) OTHER INFORMATION: - #/note= "JORAP2.12L primer"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - TCACAGAGCG TTTCTGAGAA TTAGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "AP2U primer"                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - ATGTGGGATC TAAACGACGC AC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "AP2L primer"                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - GATCTTGGTC CACGCCGAC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.1U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - AAGAGGACCA TCTCTCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.1L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - AACACTCGCT AGCTTCTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.2U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - TGGTTCAGCA GCCAACAC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "RAP2.2L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                              - - CAATGCATAG AGCTTGAGG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.4U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                              - - ACGGATTTCA CATCGGAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "RAP2.4L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                              - - CTAAGCTAGA ATCGAATCC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.5U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                              - - TACCGGTTTC GCGCGTAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/note= "RAP2.5L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                              - - CACCTTCGAA ATCAACGACC G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "RAP2.6U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                              - - TTCCCCGAAA ATGTTGGAAC TC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:82:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..25                                                           (D) OTHER INFORMATION: - #/note= "RAP2.6L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                              - - TGGGAGAGAA AAAATTGGTA GATCG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:83:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.7U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                              - - CGATGGAGAC GAAGACTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:84:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.7L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                              - - GTCGGAACCG GAGTTACC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:85:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "RAP2.8U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                              - - TCACTCAAAG GCCGAGATC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:86:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "RAP2.8L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                              - - TAACAACATC ACCGGCTCG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:87:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.9U primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                              - - GTGAAGGCTT AGGAGGAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:88:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "RAP2.9L primer"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                              - - TGCCTCATAT GAGTCAGAG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:89:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.10U primer"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                              - - TCCCGGAGCT TTTAGCCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:90:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: - #/note= "RAP2.10L primer"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                              - - CAACCCGTTC CAACGATCC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:91:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: - #/note= "RAP2.11U primer"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                              - - TTCTTCACCA GAAGCAGAGC ATG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:92:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/note= "RAP2.11L primer"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                              - - CTCCATTCAT TGCATATAGG GACG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:93:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/note= "RAP2.12U primer"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                              - - GCTTTGGTTC AGAACTCGAA CATC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:94:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/note= "RAP2.12L primer"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                              - - AGGTTGATAA ACGAACGATG CG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:95:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/note= "Primer RISZU 1"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                              - - GGAYTGTGGG AAACAAGTTT A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:96:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: - #/note= "Primer RISZU 2"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                              - - TGCAAAGTRA CACCTCTATA CTT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:97:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/note= "Primer RISZU 3"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                              - - GCATGWGCAG TGTCAAATCC A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:98:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..20                                                           (D) OTHER INFORMATION: - #/note= "Primer RIZSU 4"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                              - - GAGGAAGTTC VAAGTATAGA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:99:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                              - - Lys Lys Ser Arg                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:100:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.3 primer"                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                             - - TCATCGCCAC GATCAACC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:101:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/note= "RAP2.3 primer"                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                             - - AGCAGTCCAA TGCGACGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:102:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                             - - Trp Ala Ala Glu Ile Arg Asp                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:103:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                             - - Met Ala Asp Ser                                                         __________________________________________________________________________

What is claimed is:
 1. A method of modulating seed trait(s) in a soybeanor canola plant, the method comprising:providing a first plantcomprising a recombinant expression cassette containing an ADC nucleicacid linked to a plant promoter, the ADC nucleic acid comprisingeitheri) a nucleic acid that hybridizes under stringent conditions toSEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, ii) a nucleic acid that encodesa protein encoded by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or iii) anucleic acid amplified by primers that selectively hybridize understringent conditions to the primers GGAYTGTGGGAAACAAGTTTA (SEQ ID NO:95) and TGCAAAGTRACACCTCTATACTT (SEQ ID NO: 96) or the primersGCATGWGCAGTGTCAAATCCA (SEQ ID NO:97) and GAGGAAGTTCVAAGTATAGA (SEQ IDNO: 98); selfing the first plant or crossing the first plant with asecond plant, thereby producing a plurality of seeds; and selecting seedwith altered mass or altered carbohydrate protein or oil content.
 2. Themethod of claim 1, wherein expression of the ADC nucleic acid inhibitsexpression of an endogenous ADC gene or activity of an ADC peptide andthe step of selecting includes the step of selecting seed with increasedmass or increased carbohydrate, protein or oil content.
 3. The method ofclaim 2, wherein the ADC nucleic acid is linked to the plant promoter inthe antisense orientation.
 4. The method of claim 2, wherein the ADCnucleic acid comprises SEQ ID NO:1.
 5. The method of claim 2, whereinthe ADC nucleic acid comprises SEQ ID NO:2 or SEQ ID NO:3.
 6. The methodof claim 2, wherein the plant promoter is a constitutive promoter. 7.The method of claim 6, wherein the promoter is a CAMV 35S promoter. 8.The method of claim 2, wherein the promoter is a tissue-specificpromoter.
 9. The method of claim 8, wherein the promoter isovule-specific.
 10. A seed produced by the method of claim
 2. 11. Themethod of claim 1, wherein the plant is a soybean plant.
 12. The methodof claim 1, wherein the plant is a canola plant.
 13. The method of claim1, wherein expression of the ADC nucleic acid enhances expression of anADC nucleic acid over the wildtype level of an endogenous ADC gene orenhances ADC peptide activity over the wildtype level of an endogenousADC peptide, and the step of selecting includes the step of selectingseed with decreased mass or decreased carbohydrate, protein or oilcontent.
 14. The method of claim 13, wherein the ADC nucleic acidcomprises SEQ ID NO:1.
 15. The method of claim 13, wherein the ADCnucleic acid comprises SEQ ID NO:2 and/or SEQ ID NO:3.
 16. The method ofclaim 13, wherein the plant promoter is a constitutive promoter.
 17. Themethod of claim 16, wherein the promoter is a CAMV 35S promoter.
 18. Themethod of claim 13, wherein the promoter is a tissue-specific promoter.19. The method of claim 18, wherein the promoter is ovule-specific. 20.A seed produced by the method of claim
 13. 21. A seed comprising arecombinant expression cassette containing an ADC nucleic acid, whereinthe ADC nucleic acid comprises eitheri) a nucleic acid that hybridizesunder stringent conditions to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3,or ii) a nucleic acid that encodes a protein encoded by SEQ ID NO: 1,SEQ ID NO:2 or SEQ ID NO:3.
 22. The seed of claim 21, which is derivedfrom a canola plant.
 23. The seed of claim 21, which is derived from asoybean plant.
 24. The seed of claim 21, wherein the ADC nucleic acidcomprises SEQ ID NO:1.
 25. The seed of claim 21, wherein the ADC nucleicacid comprises SEQ ID NO:2 or SEQ ID NO:3.
 26. The seed of claim 21,wherein the ADC nucleic acid is linked to a plant promoter in anantisense orientation and the seed mass is at least about 10% greaterthan the average mass of seeds from the same plant variety which lackthe recombinant expression cassette.
 27. The seed of claim 26, whereinthe mass is at least about 20% greater than the average mass of seedsfrom the same plant variety which lack the recombinant expressioncassette.
 28. The seed of claim 26, wherein the mass is at least about50% greater than the average mass of seeds from the same plant varietywhich lack the recombinant expression cassette.
 29. The seed of claim26, wherein the oil content is increased.
 30. The seed of claim 26,wherein the protein content is increased.
 31. The seed of claim 26,wherein the carbohydrate content is increased.
 32. The seed of claim 21,wherein the ADC nucleic acid is linked to a plant promoter in the senseorientation and the seed mass is at least about 10% less than theaverage mass of seeds of the same plant variety which lack therecombinant expression cassette.
 33. The seed of claim 32, which has amass at least about 20% less than the average mass of seeds of the sameplant variety which lack the recombinant expression cassette.
 34. Theseed of claim 33, which has a mass at least about 50% less than theaverage mass of seeds of the same plant variety which lack therecombinant expression cassette.
 35. The seed of claim 32, wherein theoil content is decreased.
 36. The seed of claim 32, wherein the proteincontent is decreased.
 37. The seed of claim 32, wherein the carbohydratecontent is decreased.
 38. A transgenic plant comprising an expressioncassette containing a plant promoter operably linked to a heterologousADC polynucleotide, wherein the ADC polynucleotide comprises eitheri) anucleic acid that hybridizes under stringent conditions to SEQ ID NO:1,SEQ ID NO:2 or SEQ ID NO:3, or ii) a nucleic acid that encodes a proteinencoded by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
 39. The transgenicplant of claim 38, wherein the ADC nucleic acid comprises SEQ ID NO:1.40. The transgenic plant of claim 38, wherein the ADC nucleic acidcomprises SEQ ID NO:2 or SEQ ID NO:3.
 41. The transgenic plant of claim38, wherein the plant is a canola plant.
 42. The transgenic plant ofclaim 38, wherein the heterologous ADC polynucleotide encodes a ADCpolypeptide.
 43. The transgenic plant of claim 38, wherein theheterologous ADC polynucleotide is linked to the promoter in anantisense orientation.
 44. The transgenic plant of claim 38, wherein theplant is a soybean plant.
 45. An isolated nucleic acid moleculecomprising an expression cassette containing a plant promoter operablylinked to a heterologous ADC polynucleotide, wherein the ADCpolynucleotide comprises eitheri) a nucleic acid that hybridizes understringent conditions to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or ii)a nucleic acid that encodes a protein encoded by SEQ ID NO:1', SEQ IDNO:2 or SEQ ID NO:3.
 46. The isolated nucleic acid molecule of claim 45,wherein the ADC nucleic acid comprises SEQ ID NO:1.
 47. The isolatednucleic acid molecule of claim 45, wherein the ADC nucleic acidcomprises SEQ ID NO:2 or SEQ ID NO:3.
 48. The isolated nucleic acid ofclaim 45, wherein the heterologous ADC polynucleotide encodes a ADCpolypeptide.
 49. The isolated nucleic acid of claim 45, wherein theheterologous ADC polynucleotide is linked to the promoter in anantisense orientation.
 50. An isolated nucleic acid encoding an ADCpolypeptide, wherein the ADC nucleic acid comprises eitheri) a nucleicacid that hybridizes under stringent conditions to SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:3, or ii) a nucleic acid that encodes a proteinencoded by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
 51. The nucleic acidof claim 50, wherein the nucleic acid hybridizes to SEQ ID NO:1.
 52. Thenucleic acid of claim 51, wherein the nucleic acid comprises SEQ IDNO:1.
 53. The nucleic acid of claim 50, wherein the nucleic acidhybridizes to SEQ ID NO:2 or SEQ ID NO:3.
 54. The nucleic acid of claim53, wherein the nucleic acid comprises SEQ ID NO:2.
 55. The nucleic acidof claim 53, wherein the nucleic acid comprises SEQ ID NO:3.